US5677421A - Target proteins for eukaryotic tyrosine kinases - Google Patents

Target proteins for eukaryotic tyrosine kinases Download PDF

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US5677421A
US5677421A US08/208,887 US20888794A US5677421A US 5677421 A US5677421 A US 5677421A US 20888794 A US20888794 A US 20888794A US 5677421 A US5677421 A US 5677421A
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grb
protein
seq
amino acid
proteins
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Joseph Schlessinger
Edward Y. Skolnik
Benjamin L. Margolis
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New York University NYU
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Priority to US08/252,820 priority patent/US5889150A/en
Priority to CA002184988A priority patent/CA2184988A1/fr
Priority to PCT/US1995/003385 priority patent/WO1995024426A1/fr
Priority to EP95913755A priority patent/EP0753010A4/fr
Priority to AU21022/95A priority patent/AU2102295A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/62—Insulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Definitions

  • the invention in the field of molecular and cell biology, relates to a novel method, based on direct expression cloning, for identifying target proteins capable of binding to and/or serving as substrates for receptor or cytoplasmic tyrosine kinases.
  • the invention also relates to novel proteins identified using this method.
  • polypeptide growth factors and hormones mediate their cellular effects by interacting with cell surface receptors and soluble or cytoplasmic polypeptide containing molecules having tyrosine kinase enzymatic activity (for review, see Williams, L. T. et Cell 61:203-212 (1990); Carpenter, G. et al. J. Biol. Chem. 265:7709-7712 (1990)).
  • the interaction of these ligands with their receptors induces a series of events which include receptor dimerization and stimulation of protein tyrosine kinase activity.
  • EGFR epidermal growth factor receptor
  • PDGFR platelet-derived growth factor receptor
  • kinase activation and receptor autophosphorylation result in the physical association of the receptor with several cytoplasmic substrates (Ullrich et al., supra).
  • PLC-7 phosphatidylinositol specific phospholipase C-7
  • GAP GTPase activating protein
  • activated PDGFR was shown to tyrosine phosphorylate, and to become associated with PLC- ⁇ , GAP, and cellular tyrosine kinases such as pp60 src (Gould, K. L. et al., Molec. Cell. Biol. 8:3345-3356 (1988); Meisenhelder, J. et al., Cell 57:1109-1122 (1989); Molloy, C. J. et al., Nature 342:711-714 (1989); Kaplan, D. R. et al., Cell 61:121-133 (1990); Kazlauskas, A.
  • SH2 (src homology 2) domains appear to be the regions responsible for the association of several tyrosine kinase substrates with activated growth factor receptors. SH2 domains are conserved sequences of about 100 amino acids found in cytoplasmic non-receptor tyrosine kinases such as pp60src, PLC- ⁇ , GAP and v-crk (Marcher, B. J. et al., Nature 332:272-275 (1988); Pawson, T. Oncoqene 3:491-495 (1988)).
  • Tyrosine kinase activation and receptor autophosphorylation are prerequisites for the association between growth factor receptors and SH2 domain-containing proteins (Margolis, B. et al., Mol. Cell. Biol. 10:435-441 (1990); Kumjian et al., Proc. Natl. Acad. Sci. USA 86:8232-8239 (1989); Kazlauskas, A. et al., Science 247:1578-1581 (1990)).
  • the carboxy-terminal (C-terminal) fragment of the EGFR which contains all the known autophosphorylation sites, binds specifically to the SH2 domains of GAP and PLC-7 (see below).
  • a major site of association exists between the SH2 domain of these substrate proteins and the tyrosine phosphorylated C-terminal tail of the EGFR.
  • Target proteins which bind to activated receptors have been identified by analysis of proteins that co-immunoprecipitate with growth factor receptors, or that bind to receptors attached to immobilized matrices (Morrison, D. K. et al., Cell 58:649-657 (1989); Kazlauskas, A. et al., EMBO J. 9:3279-3286 (1990)). While the identity of some of these proteins is known, several others detected utilizing these approaches have not been fully characterized. Moreover, it is possible that rare target molecules which interact with activated receptors have not been detected due to the limited sensitivity of these techniques; the actual stoichiometry of binding may be low, and the detergent solution necessary to solubilize proteins may disrupt binding.
  • the cloning method is based on the ability of a certain class of substrates to bind specifically to the tyrosine-phosphorylated carboxy-terminus (C-terminus) of the proteins having tyrosine kinase activity.
  • Non-limiting examples include proteins that bind at least one of cytoplasmic and receptor tyrosine kinases, such as a receptor tyrosine kinase found in epidermal growth factor receptor (EGFR) (see, e.g., Example VI, below).
  • EGFR epidermal growth factor receptor
  • Another object of the present invention is to provide a method of cloning tyrosine kinase target proteins, which method important advantages over conventional cloning methods, including avoidance of the laborious and costly task of purifying potential target proteins for microsequencing analysis.
  • Another object of the present invention is to provide a method for identifying receptor target molecules having tyrosine kinase activity whose association with activation receptors could not otherwise be detected using conventional techniques.
  • Another object of the present invention is to provide for the identification of structurally or functionally related proteins which, though only weakly homologous at the nucleic acid level, are similar in their property of binding to activated receptors with tyrosine kinase activity, which latter ability is important since conventional screening methods used to identify related genes are typically based on low stringency nucleic acid hybridization. Conventional hybridization-based screening would not have been successful in cloning and identifying such tyrosine kinase target proteins of the present invention, exemplified as non limiting examples as GRB-1, GRB-2, GRB-3, GRB-4, GRB-7 or GRB-10, because of their lack of similarity at the DNA level.
  • the methods of the present invention take advantage of the discovery that the C-terminus of the EGFR protein in which the tyrosine residues are phosphorylated can bind substrates as described herein.
  • a probe is provided having at least one phosphorylated tyrosine. Such a probe can be used to detect, identify and/or purify target proteins from solutions or as part of screening of cDNA expression libraries from eukaryotic cells or tissues.
  • GRB growth factor Receptor Bound
  • target proteins are not limited to growth factor receptors.
  • GRBs of the present invention include target proteins for any eukaryotic tyrosine kinase which are provided according to the present invention.
  • CORT Cloning Of Receptor Targets
  • the method of the present invention is proposed as a novel approach having both generality and rapidity for the identification and cloning of target molecules for tyrosine kinases.
  • the present invention is thus directed to a method for detecting a target protein in solution, which is a target of a receptor or cytoplasmic tyrosine kinase, the target protein being capable of binding to at least a portion of a tyrosine-phosphorylated polypeptide of the receptor or cytoplasmic tyrosine kinase, the method comprising: (a) contacting the solution (as a cell, an extract thereof, a lysate thereof, or a supernatant thereof) with a solid phase carrier, causing the binding of the protein to the carrier to provide a carrier-bound target protein; (b) incubating the carrier-bound target protein with the tyrosine-phosphorylated polypeptide, which has been detectably labeled, allowing the polypeptide to bind to the carrier-bound protein; (c) removing materials not bound to the carrier-bound target protein; (d) detecting the presence or measuring the amount of the tyrosine-phosphorylated polypeptide bound to
  • the receptor or cytoplasmic tyrosine kinase is any eukaryotic tyrosine kinase (e.g., epidermal growth factor receptor, a platelet-derived growth factor receptor, or a fibroblast growth factor receptor), pp60 v-src , pp160 gag-abl , pp130 gag-fps , pp59 c-fyn , PDGF receptor B, CSF-1 receptor, pp150 c-fms , pp150 v-fms , EGF receptor, Insulin Receptor, IGF-1 receptor, pp68 gag-ros , PLC- ⁇ , middle t-pp60 c-src middle t-pp62 c-yes , and/or the consensus sequences EEEEEY(PO 4 )MPFIXX (SEQ ID NO:11), EEEEEY(PO 4 )VPMXX (SEQ ID NO:12), DDDDDY(PO 4 )MP
  • This method is preferably performed using a prokaryotic cell, most preferably a bacterial cell such as E. coli.
  • the cell may also be eukaryotic, such as a yeast or a mammalian cell.
  • the phosphorylated polypeptide is detectably labeled.
  • the solid phase carrier can be any material which can be used to bind a target protein for a tyrosine kinase.
  • the carrier may preferably be a nitrocellulose membrane, such as to which are transferred proteins released for lysed bacterial cells when a library is being screened.
  • the present invention also provides a method for mapping to a eukaryotic, such a mammalian, human, murine, or other eukaryotic chromosome a gene encoding a protein which is capable of binding to a tyrosine-phosphorylated polypeptide portion of a receptor or cytoplasmic tyrosine kinase molecule, the method comprising (a) infecting a host or host cells which a eukaryotic gene expression library; (b) detecting a clone expressing the protein using a method according to claim 1; (c) sequencing the DNA of the clone; and (d) mapping the sequence to a eukaryotic chromosome.
  • a eukaryotic such as a mammalian, human, murine, or other eukaryotic chromosome a gene encoding a protein which is capable of binding to a tyrosine-phosphorylated polypeptide portion of a receptor or cytoplasmic
  • the present invention is also directed to a polypeptide probe useful in the detection of the expression of a protein capable of binding to a tyrosine-phosphorylated polypeptide portion of a receptor or cytoplasmic tyrosine kinase.
  • the probe comprises an amino acid sequence derived from the tyrosine-phosphorylated portion of the receptor or cytoplasmic molecule, or a functional derivative thereof, lacks the tyrosine kinase domain, and the sequence can preferably contain at least one phosphotyrosine residue, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 phosphotyrosines.
  • the probe can preferably be detectably labeled with known labels.
  • a preferred probe has between about 10 and 250 amino acid residues, preferably 10-35, 16-30, 21-35, 15-35, or 20-40 residues.
  • a probe of the present invention is useful for detecting target proteins for receptor or cytoplasmic tyrosine kinases, including but not limited to, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), colony stimulating factor-1, (CSF-1), insulin receptor, phospholipase C- ⁇ (PLC- ⁇ ) and insul.in like growth factor-1, (IGF-1), pp60 v-src , pp160 gag-abl , pp130 gag-fps , pp59 c-fyn , PDGF receptor B, CSF-1 receptor, pp150 c-fms , pp150 v-fms , EGF receptor, insulin receptor, IGF-1 receptor, pp68 gag-ros , PLC, middle t-pp60 c-src , middle t-62 c-yes , and the consensus sequence EEEEEY(PO4)MPMXX (SEQ ID NO:
  • the present invention also includes a method for preparing the above probe, comprising (a) providing the receptor or cytoplasmic tyrosine kinase, or a recombinantly, enzymatically or synthetically produced fragment thereof, wherein the receptor or cytoplasmic tyrosine kinase, or fragment thereof, has both a tyrosine kinase domain and a tyrosine-phosphorylated domain, the tyrosine-phosphorylated domain including at least one tyrosine residue capable of being phosphorylated by the tyrosine kinase; (b) incubating the receptor or cytoplasmic tyrosine kinase, or fragment, with detectably labeled adenosine triphosphate under conditions permitting phosphorylation of the tyrosine residue, causing phosphorylation of the tyrosine residue thereby producing the probe.
  • the method further includes the step of: (c) additionally treating the phosphorylated receptor or cytoplasmic tyrosine kinase molecule with an agent capable of cleaving the molecule between the tyrosine kinase domain and the tyrosine-phosphorylated domain.
  • a preferred cleaving agent is cyanogen bromide.
  • the above method involves a genetically engineered receptor-like derivative which is a polypeptide encoded by a DNA molecule comprising a DNA sequence encoding tyrosine kinase, linked to a DNA sequence encoding a selective. enzymatic cleavage site, linked to a DNA sequence encoding the tyrosine-phosphorylated domain, and wherein the agent is an enzyme capable of cleaving at this cleavage site.
  • Preferred enzymes are Factor Xa and thrombin.
  • the present invention is also directed to GRB proteind of at least 10 amino acids, including any range or value up to their entire native or mature length.
  • the present invention in one embodiment provides a protein, GRB-1, having an amino acid sequence substantially corresponding to an amino acid sequence shown in FIG. 4 (SEQ ID NO:2).
  • the invention also includes polypeptides having an amino acid sequence substantially corresponding to an amino acid sequence of a protein, GRB-2, which includes the amino acid sequence shown in FIG. 26A-26C ((SEQ ID NO:6).
  • the invention also includes polypeptides having an amino acid sequence substantially corresponding to an amino acid sequence of a protein, GRB-3, which includes the amino acid sequence shown in FIG. 34A-34C (SEQ ID NO:4).
  • the invention also includes polypeptides having an amino acid sequence substantially corresponding to an amino acid sequence of a protein, GRB-4, which includes the amino acid sequence shown in FIG. 35A-35B (SEQ ID NO:8).
  • the invention also includes polypeptides having an amino acid sequence substantially corresponding to an amino acid sequence of a protein, GRB-7, which includes the amino acid sequence shown in FIG. 36A-36G (SEQ ID NO:10).
  • the invention also includes polypeptides having an amino acid sequence substantially corresponding to an amino acid sequence of a protein, GRB-10, which includes the amino acid sequence shown in FIG. 38 (SEQ ID NO:49).
  • the invention is also directed to a DNA or RNA molecule encoding a polypeptide having at least a 10 amino acid sequence substantially corresponding to the amino acid sequence of at least one of GRB-1, GRB-2, GRB-3, GRB-4 GRB-7 and GRB-10. Included are DNA molecules encoding functional derivatives of these proteins. When the DNA molecule naturally occurs, it is substantially free of the nucleotide sequences with which it is natively associated.
  • the DNA molecules of this invention may be expression vehicles, such as plasmids. Also provided is a host transformed with each of the above DNA molecules.
  • the present invention also includes a process for preparing a target protein substantially corresponding to the amino acid sequence GRB-1, GRB-2, GRB-3, GRB-4, GRB-7 or GRB-10 protein, comprising: (a) culturing a host comprising a recombinant nucleic acid having a nucleotide sequence encoding the target protein under culturing conditions such that the target protein is expressed in recoverable amounts; and (b) recovering the protein from the culture.
  • FIG. 1 is a filter blot pattern showing that the carboxy-terminus of the EGFR interacts with GAP-SH2 immobilized on nitrocellulose filters.
  • Bacterially-expressed trpE/GAP-SH2 fusion protein or trpE as a control was spotted at various concentrations onto nitrocellulose filters. The filters were hybridized overnight with ( 32 P)-labelled C-terminal domain of the EGFR. Autoradiography was for 2 hours.
  • FIG. 2 is a schematic diagram depicting the method of cloning of receptor or cytoplasmic tyrosine kinase targets (CORT).
  • C-terminal domain of the EGFR is phosphorylated with radiolabelled phosphorous.
  • Lambda gtll library was plated at a density of 4 ⁇ 10 4 plaques per 150 ml plate. The plaques were overlaid with IPTG-impregnated nitrocellulose filters for 12 hours, after which the plaques were transferred to nitrocellulose and incubated with the labelled probe. Positive colonies are then selected for further analysis.
  • FIG. 3A-B shows autoradiograms of phage expressing GRB-1 protein.
  • FIG. 3A shows a primary screen demonstrating one positive signal (arrow) out of 40,000 phage plated.
  • FIG. 3B shows a plaque purification of phage expressing GRB-1. All plaques bound to the ( 32 P)-labelled C-terminal domain of the EGFR.
  • FIG. 4A to 4I shows the DNA sequence and corresponding amino acid sequence of GRB-1 (SEQ ID NO:1-2).
  • the protein in one form has 724 amino acid residues.
  • FIG. 5 compares the sequences of the SH2 domains of GRB-1 with other proteins with similar motifs.
  • N and C refer to N-Terminal and C-terminal SH2 domains respectively.
  • FIG. 5B shows a similar comparison of the SH3 domain of GRB-1.
  • FIG. 6 is a schematic diagram comparing the structural organization of the SH2 and SH3 domains.
  • the scheme includes known proteins containing SH2 and SH3 domains, such as c-src, v-crk, PLC- ⁇ , GAP1 and GRB-1.
  • FIG. 7 is a Northern blot of monkey mRNA with GRB-1 probe. 5 ⁇ g of poly (A)+MRNA, obtained from various monkey tissue, was electrophoresed on 1.2%/2.2M agarose-formaldehyde gel. The blot was hybridized with a ( 32 P)-nick translated DNA probe corresponding to the insert from clone ki4.
  • FIG. 8 is a gel pattern showing that antibodies to GRB-1 immunoprecipitate a protein of 85 kDa from biosynthetically labelled cells.
  • Cells were metabolically labelled with ( 35 S)methionine, after which lysates were prepared and immunoprecipitated with either immune (I) or preimmune (P) serum.
  • the immunoprecipitated protein was separated on a 8% SDS/PAGE. Autoradiography was performed overnight.
  • Cell lines used include human glioblastoma cell line, U1242, rat bladder carcinoma cell line, NBT-II and NIH-3T3 cells.
  • FIG. 9A and 9B depicts several wild-type and mutant proteins used in the studies.
  • EGF receptor constructs with their known or predicted autophosphorylation sites. Wild-type (W. T.), Kinase negative (K721A), and carboxy-terminal deletion (CD126), were immunoprecipitated from previously described transfected NIH373 cells expressing -300,000 EGF receptors.
  • EGFR-C represents a deletion mutant containing the cytoplasmic domain of the EGF receptor produced by baculovirus-infected SF9 cells.
  • B Structure of PLC- ⁇ and trpE/GAP SH2 proteins indicating location of the SH2 and SH3 domains and PLC- ⁇ tyrosine phosphorylation sites.
  • FIG. 10A-10B is a gel pattern showing association of PLC- ⁇ with EGFR mutants.
  • Wild-type (HER14), carboxy-terminal deletion (DC126), or kinase-negative (K721A) EGFR were immunoprecipitated with anti-EGFR mAb108.
  • Receptors were autophosphorylated with ( ⁇ - 32 P-ATP.
  • Concomitantly EGFR-C was added to protein A-Sepharose beads alone or to immunoprecipitated K721A receptors either with or without ATP. After further washes to remove ATP, lysate from 15 ⁇ 10 6 PLC-T overexpressing 3T-P1 cells was added and mixed for 90 min at 4° C.
  • FIG. 11 is a gel pattern showing that phosphorylation of PLC- ⁇ reduces its binding to the EGF receptor.
  • Full length EGFR was immunoprecipitated with mAb108, and allowed to autophosphorylate. Lysate from PLC- ⁇ overexpressing 3T-P1 cells was added and mixed for 90 min at 4° C. After binding, ATP was added to one half of the samples allowing the PLC- ⁇ molecules to be phosphorylated by the EGF receptor. SDS-PAGE sample buffer was then added to one half of the EGFR-PLC-T complexes (NO WASH, left panel) and directly loaded onto the 6% gel. The other half was washed three times with HNTG and then loaded on the gel (WASH, right panel.
  • FIGS. 12A and 12B are representations of a gel pattern showing binding of EGFR-C to trpE proteins.
  • EGFR-C 0.5 ⁇ g
  • MnCl 2 alone or MnCl 2 and ATP were then added to facilitate autophosphorylation of TrpE or trpE/GAP SH2 (approximately 2 ⁇ g).
  • the immunoprecipitates were separated on a 10% SDS-gel, transferred to nitrocellulose and immunoblotting was performed with anti-trpE. For comparison, about 0.1 ⁇ g of trpE or trpE/GAP SH2 lysate was loaded directly on to the gel (right panel of A).
  • trpE or trpE/GAP SH2 was immunoprecipitated with anti-trpE antibodies and washed.
  • Phosphorylated or non-phosphorylated EGFR-C (0.5 ⁇ g) was then added and allowed to bind as above. After washing, samples were separated on a 10% gel, transferred to nitrocellulose and probed with antibody C. The two samples on the right represent 0.5 ⁇ g of phosphorylated and non-phosphorylated kinase loaded directly onto the gel (exposure time: 2 h).
  • FIGS. 13A and 13B are representations of a gel pattern showing binding of trpE/GAP SH2 to wild-type and mutant EGFR.
  • wild-type receptor (HER14) or the carboxy-terminal deletion CD126 receptor were immunoprecipitated with mAb 108.
  • MnCl 2 alone or MnCl 2 and ATP were then added to the autophosphorylated half of the receptor-containing samples.
  • One set of CD126 was also cross-phosphorylated with 0.5 ⁇ g of EGFR-C.
  • TrpE/GAP SH2 was then added for 90 min at 4° C. and, after three more washes, loaded onto SDS-PAGE.
  • blots were probed with anti-trpE (left panel), anti-EGFR RK2 (center panel), or anti-PTyr (right panel).
  • RK2 and anti-PTyr are both 1/8 of the total sample and were separated on 7% SDS-PAGE. The remaining sample was loaded on a 10% gel for the anti-trpE blot (exposure time 14 h).
  • lysates from NIH3T3 2.2 cells containing no EGFR (3T3) or from cells with kinase-negative receptors (K21A) were immunoprecipitated with mAb108.
  • mAb108 kinase-negative receptors
  • 0.5 ⁇ g of EGFR-C was added and then MnCl 2 alone or MnCl 2 and ATP.
  • trpE/GAP SH2 was added and samples prepared and immunoblotted as in (A) (exposure time 19 h).
  • FIG. 14 is a gel pattern showing binding of PLC- ⁇ and trpE/GAP SH2 to the CNBr cleaved C-terminal fragment of EGFR.
  • EGFR-C (10 ⁇ g) was incubated in a Centricoh 30 in 20 mMHEPES, pH 7.5 with 100 ⁇ g BSA as a carrier protein. The phosphorylated and non-phosphorylated EGFR-C were then each divided in two with one half being stored in buffer while the other half was cleaved with CNBr. The four samples either with or without ATP, and with or without CNBr were then each brought up in 500 ⁇ l 1% Triton X-100 lysis buffer, split in two, and immunoprecipitated with anti-C antibody.
  • FIG. 15 is a gel pattern showing binding of the tyrosine phosphorylated C-terminal EGFR fragment to trpE/GAP SH2 but not to trpE.
  • EGFR-C (5 ⁇ g) was autophosphorylated by the addition of ( ⁇ -32P)ATP. The phosphorylated EGFR-C was concentrated in a Centricoh 30, and then cleaved with CNBr in 70% formic acid. One half of the sample (350,000 c.p.m.) was allowed to bind to trpE or trpE/GAP SH2 as in FIG. 12B, washed and run on a 10% SDS-gel.
  • B Binding of phosphorylated CNBr cleaved EGFR-C to trpE GAP SH2
  • C 3000 c.p.m. of CNBr-cleaved EGFR-C
  • D for comparison 3000 c.p.m. of cleaved EGFR-C (exposure time 20 h).
  • EGFR 984/1186 indicates the sequence of the tyrosine autophosphorylated fragment generated by CNBr.
  • FIGS. 16A-16D shows the partial nucleotide sequence (SEQ ID NO:32) and predicted amino acid sequences (SEQ ID NOS:33-38) of GRB-2.
  • FIG. 17 is a comparison of sequence homology of arian crk (SEQ ID NO:39) to GRB-3 (SEQ ID NO:4) with dots indicating homologous amino acids.
  • FIG. 18 is a protein sequence of nck (SEQ ID NO:40) compared to that of GRB-4 (SEQ ID NO:8) for amino acid sequence homology.
  • FIG. 19 is a GRB-7 (SEQ ID NO:10) protein sequence.
  • FIG. 20 is a schematic representation of GRB-7 to include the proline rich, P2B2, rasGAP and SH2 domain homology.
  • FIG. 21 is a comparison of a GRB-7 amino acid sequences (SEQ ID NO:10) with SH2 domains from arian c-src (SEQ ID NO:19), human PLC- ⁇ 1(SEQ ID NO:22), GRB-1/p85(SEQ ID NO:17), mouse fyn (SEQ ID NO:41), GRB-3 (SEQ ID NO:4) and GRB-4(SEQ ID NO:8).
  • FIG. 22 is a comparison of a GRB-7 amino acid sequence (SEQ ID NO:10) with rasGAP (SEQ ID NO:42).
  • FIG. 23 is a comparison of a GRB-7 amino acid sequence (SEQ ID NO:10)with P2B2(SEQ ID NO:43).
  • FIG. 24 is a representation of a Northern blot analysis of GRB-7 mRNA.
  • FIG. 25 is a comparison of binding of the phosphorylated EGFR carboxy-terminus to PLC-g fragments expressed in a kgtll or T7 polymerase based library.
  • FIG. 26A-26C include a cbNA (SEQ ID NO:5) and protein sequence (SEQ ID NO:6) of GRB2 clone 10-53, with '5 and '3 untranslated flanking sequences; SH2 (thick line) and SH3 (thin lines) domains are indicated.
  • FIG. 26D is a schematic representation of the overall domain structure of GRB2.
  • N and C refer to N-terminal and C-terminal domains, respectively.
  • the one letter code is used to indicate amino acid residues. Bold letters identify those positions where the same or a conservative amino acid substitution is present at that position.
  • Compared are PLC%l, GAP, v-src, v-abl, v-crk and p85.
  • the SH2 domain of GRB2 is most similar to the SH2 domain of v-fgr (43% similarity) and the N-terminal SH3 domain is most similar to the SH3 domain of human ray (48% similarity).
  • FIGS. 27A-27B show the analysis of expression of GRB2 in various murine tissues and cell lines.
  • 27A shows a Northern analysis in murine tissues, with tissue of origin as indicated, with 20 ⁇ g total RNA loaded per lane.
  • the sizes of the GRB2 transcripts are 3.8 kb and 1.5 kb.
  • FIG. 27B shows immunoprecipitation of GRB2 from ( 35 S)methionine labeled HER14 lysates with preimmune (lane 1) and immune GRB2 antiserum (Ab50) (lane 2). Immunoblot analysis of GRB2 from lysates of HER14 cells with Ab86 (lane 3). Molecular weight markers (sized in kDa) are indicated. Arrow indicates band corresponding to GRB2 protein. Exposure times are 24 hours.
  • FIG. 28 shows the association of endogenous GRB2 with EGFR in HER14 cells.
  • HER14 cells mock treated (lanes 1, 3, 5) or EGF treated (lanes 2, 4, 6) were lysed and immunoprecipitated with anti-EGF receptor antibodies (mAb 108), subjected to SDS-PAGE, and after transfer to nitrocellulose, blotted with polyclonal anti-EGFR antibodies (Anti-C) (lanes 1 and 2), anti-phosphotyrosine antibodies (lanes 3 and 4), or anti-GRB2 antibodies (Ab86) (lanes 5 and 6).
  • the immunoblots were labeled with 125 I-protein A followed by autoradiography at -70° C.
  • Anti-GRB2 blot were exposed for 24 hrs.
  • Anti-EGFR and antiP-tyr blots were exposed for 16 hrs. The positions of molecular weight markers (sized in kDa) are indicated.
  • FIG. 29 is a schematic representation of GRB2-GST fusion proteins.
  • Gluthatione-S-transferase fusion proteins of full size GRB2 and various regions of GRB2 were generated and purified by affinity chromatography utilizing glutathione agarose beads, as described in methods. Shown are the SH2 domain of GRB2 (SH2), the amino terminal SH3 (N-SH3), carboxy terminal SH3 (C-SH3), the amino terminal SH3 and SH2 domains (N-SH3 SH2), and the SH domain with the carboxy terminal SH3 domain (SH2 C-SH3). GST region of fusion proteins is not shown.
  • FIG. 30 represents the binding of GST-GRB2 fusion proteins to activated growth factor receptors in vitro. Binding of fusion proteins to the tyrosine phosphorylated proteins (lanes 1 through 6) and EGFR (lanes 7 through 10) in control and EGF stimulated HER14 cell lysates, and tyrosine phosphorylated proteins in control and PDGF stimulated lysates (lanes 11 through 14). Lysates were incubated with equal amounts of fusion proteins immobilized on glutathione-agarose beads.
  • Bound proteins were washed, subjected to SDS-PAGE and immunoblotted with antiphosphotyrosine (lanes 1 through 6, 11 through 14)) or anti EGF-receptor (lanes 7 through 10) antibodies.
  • the immunoblots were labelled with proteins a followed by autoradiography at -70° C. exposure time 16 hrs. The positions of the molecular weight markers are indicated (sizes in kDA).
  • FIG. 31 shows data representing the lack of significant phosphorylation of GRB2 in HER14 cells following stimulation with EGF.
  • 32 P orthophosphate (lanes 1 through 4) or (35S) methionine (lanes 5 through 8) metabolically labeled HER14 cells were lysed following mocked EGF treatment.
  • the precleared lysates were immunoprecipitated with either preimmune or anti-GRB2 antibodies (Ab50), and subjected to SDS-PAGE and autoradiography. Two hour (32P) and two day ( 35 S) exposure times are shown.
  • the position of GRB2 and the co-immunoprecipitating 55 kDa phosphoprotein are marked with arrows.
  • FIG. 32 presents the alignment of amino acid sequences of GRB2 (SEQ ID NO:6) and sem-5 (SEQ ID NO:47) (single letter code). Boxes surround the SH2 and SH3, domains, as indicated. Bold capital letters indicate identical amino acids, capital letter indicate conservative substitutions.
  • FIG. 33 is a representation showing a model for the interaction between EGF receptor and GRB2 and their C. elegans counterparts.
  • Tyrosine autophosphorylated EGFR (or let-23) binds to the SH domain of GRB2 (or sem-5).
  • Ras (or let-60) acts downstream leading to either cell proliferation or vulval development.
  • FIG. 34A-34C is a cDNA (SEQ ID NO:3) and protein sequence (SEQ ID NO:4) of GRB-3.
  • FIG. 35A-35B is a cDNA (SEQ ID NO:7) and protein (SEQ ID NO:8) sequence of GRB-4.
  • FIG. 36A-36G is a cDNA (SEQ ID NO:9) and protein (SEQ ID NO:10) sequence of GRB-7.
  • FIG. 37A-37C cDNA sequence including the coding sequence of GRB-10 (SEQ ID NO:48).
  • SEQ ID NO:48 A partial clone encompassing GRB-10 nucleotides 1950 to 2340 and encoding the GRB-10 SH2 domain was isolated by screening a randomly primed ⁇ EX1ox library with the phosphorylated carboxyterminal tail of the EGF-Receptor. This probe was used to isolate the GRB-10 cDNA which encoded the full length protein using the CORT technique.
  • FIG. 38 Deduced protein sequence of GRB-10 (SEQ ID NO:49).
  • FIG. 39A to 39E GRB-10 cDNA (SEQ ID NO:48) and protein sequence (SEQ ID NO:49).
  • FIG. 40A-40B Alignment of the protein sequence of GRB-7 (SEQ ID NO:10) and GRB-10(SEQ ID NO:49).
  • the GRB-7 (Margolis et al. 1992, Proc. Natl. Acad. Sci. USA 89:8894-8898) and GRB-10 protein sequences were aligned using the BESTFIT program of the Wisconsin Genetics Group Sequence Analysis Software (GCG) (Devereux et al., 1984, Nucleic Acids Res. 12:387-395). Identity is indicated by the vertical lines.
  • FIG. 41 Schematic representation of the alignment of GRB-7, GRB-10 and FLOE9.6.
  • GRB-7 and GRB-10 both display SH2 domains in their carboxyterminus.
  • FIG. 42 Alignment of the GRB-10 SH2 domain (Portion of SEQ ID NO:49) with those found in GRB-7(Portion of SEQ ID NO:10), GRB-2 (portion of SEQ ID NO:6) and c-Src (SEQ ID NO:50). SH2 domains were aligned using the GCG programs LINEUP, PILEUP and PRETTY (Devereux et al., 1984, Nucleic Acids Res. 12:387-395).
  • FIG. 43 Alignment of the central domains of GRB-7(portion of SEQ ID NO:10), GRB-10 (portion of SEQ ID NO:49) and FLOE9.6(SEQ ID NO:51). Alignment was performed using the GCG programs LINEUP, PILEUP and PRETTY with capital letters indicating identity or conservative substitution.
  • FLOE9.6 represents a putative gene derived from genomic sequence of C. Elegans using the program GENEFINDER. The FLOE9.6 sequences were deposited into Genbank by the C. Elegans Sequencing Consortium, Genbank accession number L10986 (Sulston et al., 1992, Nature 356:37-41).
  • FIG. 44 Northern blot of GRB-10 Poly(A)+ RNA.
  • Human umbilical vein endothelial cells Huvec: human umbilical vein endothelial cells; Jurkat: human T cell leukemia cell line).
  • One embodiment of the present invention is to provide a novel expression/cloning system for the rapid cloning of target proteins which bind tyrosine kinase proteins. which are present intracellularly and in cell receptors of eukaryotes.
  • the cloning method is based on the discovery that certain class of substrates can bind specifically to the phosphorylated domain of proteins having tyrosine kinase activity.
  • novel probes and methods using such probes for rapid expression cloning of DNA encoding proteins which have the characteristic of binding to the tyrosine-phosphorylated portion, such as the C-terminus, of a receptor tyrosine kinase molecule, which molecule is present in the cytoplasm or in cell receptors of eukaryotic receptors.
  • eukaryote or "eurkaryotic” is intended any organism considered to have the attributes of a eukaryote, including a cell nucleus, mitochondria, chromosomes, etc., which are attributes which do not occur in bacteria, blue-green algae. or viruses.
  • eukaryoees include yeast, fungi, insects, plants, mammals, birds, reptiles, amphibians. Mammals include, but are not limited to, humans, mice, rats, rabbits, cows, pigs, goats, sheep, horses, cats, dogs, etc.
  • Expression cloning is a method wherein the DNA being cloned encodes a protein which is expressed from a cloned library from a cell known or expected to have the desired protein.
  • the desired DNA typically in the form of a cDNA library, is detected by means of its exprebsion and/or direct detection of the protein which it encodes.
  • Expression cloning systems and library cloning are well-known in the art (see: Sambrook, J. et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), and Ausubel et al, eds. (Current Protocols in Molecular Biology Wiley interscience, NY (1987, 1992)), which references are hereby entirely incorporated by reference).
  • the protein is expressed according to known method steps from a library and the expressed protein, released from the cell it is expressed in is transferred to a solid carrier or support, such as a nitrocellulose filter as a non-limiting example, and detected using a detectable label for the expressed protein by known method steps.
  • a solid carrier or support such as a nitrocellulose filter as a non-limiting example
  • polypeptide probe target protein can be detectably labeled is by providing peptide probes or anti-target protein antibodies and linking the peptide probes or antibodies to an enzymefor use in an enzyme immunoassay (EIA).
  • EIA enzyme immunoassay
  • This enzyme when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means.
  • Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection may additionally be accomplished using any of a variety of other immunoassays or detectably labeled peptide probes.
  • immunoassays for example, by radioactively labeling the peptide probes, anti-target protein antibodies or antibody fragments, such that the labeled target protein may also be detected through the use of a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • a good description of RIA may be found in Laboratory Techniques and Bio-chemistry in Molecular Biology, by Work, T. S., et al., North Eolland Publishing Company, New York (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by T. Chard, incorporated by reference herein.
  • a radioactive isotope such as 32 P, 35 S, 12 C or 3 H, can be detected by such means as the use of a gamma counter, a liquid scintillation counter or by autoradiography.
  • fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • fluorescent probes are well known or commercially available, such as from Molecular Probes, inc., Eugene Oreg.
  • the peptide probe or anti-target protein antibody can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others. of the lanthanide series. These metals can be attached to the peptide probe or anti-target protein antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the peptide probe or anti-target protein antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged peptide probe or anti-target protein antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the peptide probe or anti-target protein antibody of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic peptide probe or anti-target protein antibody increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent peptide probe or anti-target protein antibody is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • the expression cloning method of the present invention for detecting and cloning a target protein for tyrosine kinase cytoplasmic or receptor protein may be used for detecting such target proteins from any eukaryotic cell source.
  • certain target molecules bind to the tyrosine phosphorylated portion of PDGFR and the colony stimulating factor-1 (CSF-1) (Coughlin, S. R. et al., Science 243:1191-1194 (1989); Kazlauskas, A. et al., Cell 58:1121-1133 (1989); Shurtleff, S. A. et al., EMBO J. 9:2415-2421 (1990); and Reedjik, M. et.
  • CSF-1 colony stimulating factor-1
  • tyrosine phosphorylation occurs in a kinase insert domain, rather than in the C-terminal domain as is the case with the EGFR. Therefore, specific polypeptide probes in the range of 10-250, such as 10-20, 20-30, 40-50, 70-100, or 100-200, amino acids utilizing the kinase insert domain, or a portion thereof as defined herein, and cytoplasmic or receptor or PDGFR or CSF-1 receptor can be similarly used for expression cloning.
  • Similar probes can also be constructed for the fibroblast growth factor (FGF) receptor (which is tyrosine phosphorylated in the C-terminal domain) or the HER 2/neu receptor, both of the which are also able to interact with SH2 containing proteins such as PLC- ⁇ .
  • FGF fibroblast growth factor
  • HER 2/neu receptor both of the which are also able to interact with SH2 containing proteins such as PLC- ⁇ .
  • SH2 containing proteins such as PLC- ⁇ .
  • tyrosine phosphorylation occurs in the kinase domain itself.
  • any tyrosine kinase protein or fragment thereof of 10-250 amino acids can be used to bind a target protein in solution which is contacted to the tyrosine kinase protein bound or associated with a carrier or support.
  • the carrier or support can be any known material that associates with a tyrosine kinase or fragment thereof, such that, once the target protein is bound, the non-bound material can be removed from the carrier without dissociated the tyrosine kinase bound to the target protein.
  • the tyrosine kinase protein is used as a protein probe to bind target proteins.
  • a polypeptide of 10-250 amino acids, corresponding to at least a phosphorylation domain of the tyrosine kinase; or corresponding to a consensus sequence of a class or group of tyrosine kinases, can be used as the protein or polypeptide probe and may be detectably labeled.
  • the present invention recognizes the common features of all these structures, the presence of one or more phosphotyrosine residues, and the ability of certain cellular proteins to bind on the basis of affinity to a polypeptide containing one or more phosphotyrosines. While reference will generally be made below to a probe which is a C-terminal domain, with reference to the EGFR, this language is not intended to be limiting and is intended to include all of the other alternative tyrosine-phosphorylated domains discussed above.
  • the methods and approach of the present invention can be applied to the cloning and identification of all target molecules which are capable of interacting in a specific manner with tyrosine phosphorylated polypeptides, such as cytoplasmic tyrosine kinases or the activated phosphorylated receptors described herein.
  • tyrosine phosphorylated polypeptides such as cytoplasmic tyrosine kinases or the activated phosphorylated receptors described herein.
  • Additional proteins which bind to tyrosine-phosphorylated sequences such as the tyrosine-specific phosphatases, e.g., R-PTPases (Sap, J. et al., Proc. Natl. Acad. Sci. USA 87:6112-6116 (1990); Kaplan, R. et al., Proc. Natl. Acad. Sci.
  • USA 87:7000-7004 (1990) may also be use according to a method of the present invention.
  • the methods are also applicable in the cloning and identification of proteins which bind to phosphorylated serine/threonine residues, as with serine/threonine-specific phosphatases as a non-limiting example.
  • a polypeptide or protein probe of the present invention allows the rapid cloning of DNA and identification of the encoded proteins from eukaryotic DNA or RNA libraries, such as a gene expression library.
  • the method is particularly useful with a bacteriophage lambda gtll library or a T7 library.
  • a eukaryotic library screening a human fetal brain lambda gtll expression library has permitted the present inventors to clone several target protein genes and to characterize the proteins they encode.
  • GRB-1 was fully DNA sequenced (SEQ ID NO:1) and found to encode novel human protein with an amino acid sequence as shown in FIG.
  • GRB-2 DNA (FIG. 26A-26C) (SEQ ID NO:5) also contains unique SH2 and SH3 domains in the amino acid sequence, (FIG. 26A-26C) (SEQ ID NO:6).
  • GRB-3 DNA (SEQ ID NO:3) was also sequenced (FIG. 34A-34C) and the GRB-3 amino acid sequence (SEQ ID NO:4).
  • GRB-4 DNA (SEQ ID NO:7) (FIG. 35A-35B) encoded a protein composed of three SH3 domains and one SH2 domain having the GRB-4 amino acid sequence (SEQ ID NO:8).
  • FIG. 36A-36G A schematic representation of GRB-7 is displayed in FIG. 20 depicting the regions of similarity to known proteins.
  • the GRB-7 protein is 535 amino acids in length (FIG. 36A-36G) (SEQ ID NO:10) and has one SH2 domain at its extreme carboxy-terminus.
  • the SH2 domain of GRB-7 is compared to other SH2 domains including mouse fyn, human PLC- ⁇ 1 and the crk and nck-like proteins of the present invention.
  • amino acids 242 to 339 of GRB-7 showed similarity to a sequence from the central region of ras GAP (21). Over this region of 91 amino acids from ras GAP, GRB-7 has 26% identity and 42% similarity allowing for conservative substitutions (FIG. 22). This region of ras GAP lies between the SH2/SH3 domains and the GTPase activating carboxy terminal region and has not been assigned a specific function (Martin et al Science 255:192 (1992)). The amino-terminal sequence of GRB-7 was found to be proline rich and thus has similarity to many other proline rich proteins.
  • GRB-7 does have an extended region of limited similarity to the catalytic domain of protein phosphatase 2B (Guerini and Klee, Proc. Natl. Acad. Sci. USA 87:6112 (1990)) including this proline rich region (FIG. 23) but no significant similarity was found to other serine/threonine phosphatase such as protein phosphatase 1 or 2A.
  • FIG. 25 A northern blot of GRB-7 in mouse tissues is presented in FIG. 25. Oligo dt selected mRNA was probed with GRB-7 cDNA using known methods. See Ausubel et al eds., Current Protocols in Molecular Biology, Wiley Interscience, New York, (1987, 1992) and Sap et al Proc. Natl. Acad. Sci. USA 87:6112 (1990), which are entirely incorporated herein by reference. The highest signal was detected in liver and kidney, but a signal was also detected in ovary and testes. On longer exposure, a weak signal was detectable in lung but not in heart, muscle, spleen or brain. The major transcript was seen at 2.4 kb which closely corresponds to the longest cDNA clone obtained.
  • GRB-7 represents another novel gene cloned using the CORT technology, according to the present invention. It belongs to a relatively rare group of proteins with SH2 domains but no SH3 domains including the fps tyrosine kinase, (I. Sadowski, J.C. Stone and T. Pawson, Mol. Cell. Biol. 6:4396 (1986)), protein tyrosine phosphatase 1C (Shen et al Nature(Lond.) 352:736 (1991)) and possibly tensin (Davis et al., Science 252:712 (1991)) .
  • fps tyrosine kinase I. Sadowski, J.C. Stone and T. Pawson, Mol. Cell. Biol. 6:4396 (1986)
  • protein tyrosine phosphatase 1C Shen et al Nature(Lond.) 352:736 (1991)
  • possibly tensin Davis et al
  • CORT methodology of the present invention provides proteins that interact with the EGFR and lie downstream of the EGFR signalling pathway.
  • in vitro associations between SH2 domain and tyrosine phosphorylated proteins correlate with interactions in living cells (McGlade et al., Mol. Cell. Biol. 12:991 (1992)).
  • CORT methodology of the present invention is therefore expected to yield commercially important downstream signalling components of cytoplasmic tyrosine kinase target proteins, as well as growth factor receptors, as demonstrated by the finding that the C. elegans gene sem-5 is the homolog of human GRB-2.
  • Sem-5 is crucial for vulval development, a process that requires the activity of let-23, an EGFR like tyrosine kinase. Accordingly, it is expected that sem-5 lies downstream of the activated let-23, and that GRB-2 serves a similar crucial function in EGFR signalling.
  • CORT methodology of the present invention can also be used to identify new SH2 proteins that interact with the EGFR. Seven different exemplary SH2 domain proteins are expected to have important signalling functions. With the use of the T7 polymerase based library, this methodology may be more easily applied, due to relatively higher levels of expressions which increase detectability, to any eukaryotic cytoplasmic or receptor tyrosine kinase proteins, such as growth factor receptor systems. Hence such a method of the present invention can also be used to clone other novel SH2 domain proteins using other growth factor receptor tyrosine kinases, including the use of T7 polymerase based libraries, by performing expression/cloning. techniques involving % protein-protein interactions and DNA binding proteins.
  • SH2 domains such as in the GAP and PLC- ⁇ proteins, are responsible for the association of these proteins with the phosphorylated C-terminus of the EGFR (see Example VI, below).
  • one function of SH2 domains is to juxtapose the intracellular portion of receptor tyrosine kinase molecules with their substrates to facilitate efficient tyrosine phosphorylation.
  • GRB-1 Detailed analysis of one of the cDNA clones of the present invention, identified using methods of the present invention, reveals a novel sequence containing two SH2 domains and one SH3 domain. This protein is expressed in various tissues and cell lines. its predicted molecular weight, 85 kDa, is consistent with its migration on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
  • cytoplasmic tyrosine kinase is meant a soluble form of protein or polypeptide having tyrosine kinase which can be found in the intracellular portion of a cell.
  • receptor tyrosine kinase is intended a transmembrane protein having an extracellular receptor domain, and one or more intracellular domains, including at least one extracellular or intracellular domain having tyrosine kinase enzymatic activity. Additional intracellular domains may have sequence homology to SH2.
  • target proteins proteins which interact with, and which may be phosphorylated by, tyrosine kinases are referred to as "target" proteins for these kinases, as distinguished from the "ligands" for these receptors, which bind to the kinase.
  • an expression cloning method is performed directly on a gene expression library, such as lambda gtll or T7 expression library.
  • the DNA is human cDNA. More preferably, the DNA is human fetal brain DNA.
  • a source as the starting material for the cloning of human genes has a great advantage over the alternative .known means, in which a large amount of tissue is taken, and antibodies produced, or the protein purified and partially sequenced, and oligonucleotide probes are then prepared from this sequence and used to screen a genomic DNA or cDNA library. The advantage of bypassing these steps is of most relevance in the case of human genes, since tissue is generally not available in large quantities, with the exception of placenta.
  • the expression library may be screened in a single step.
  • the lambda plaques are blotted onto a solid carrier, preferably nitrocellulose, allowing the transfer of library DNA-encoded proteins which are expressed in the infected bacteria and transferred to the carrier.
  • This carrier is then incubated with the probe of the present invention, as described herein.
  • the probe is allowed to bind to proteins which have the capability of binding to the tyrosine-phosphorylated polypeptide.
  • an appropriate detection system is used to identify the plaques containing the protein of interest.
  • the phage in these plaques are then selected, and the DNA inserts can then be re-cloned, excised and placed into other vectors, used for large scale expression of the protein, and the like, according to known method steps.
  • Solid phase carrier Materials of which solid phase carrier can be made include, but are not limited to, nitrocellulose, cellulose, paper, substituted polystyrenes, acrylonitriles, polycarbonate, polypetene, or silicone oxide.
  • the probe of the present invention is a tyrosine-phosphorylated polypeptide molecule derived from the C-terminal domain of a cytoplasmic or receptor tyrosine kinase.
  • the polypeptide can have between about 10 and about 250 amino acids in length.
  • the probe can be a phosphorylated native sequence or a functional derivative thereof (defined below).
  • tyrosine kinase domain present on the tyrosine kinase molecule to autophosphorylate the C-terminal region at between 1 and 5 tyrosine residues.
  • Known methods and conditions (described in detail in Example I) are used to phosphorylate the tyrosine residues.
  • a preferred substrate is detectably labeled substrate such as ( ⁇ -P 32 -adenosine triphosphate).
  • the source of tyrosine molecule used as the source material to make the probe can include molecules chemically purified from tissues or cells, or molecules produced recombinant DNA methods.
  • a native cytoplasmic or receptor tyrosine kinase may be produced, or alternatively, a tyrosine kinase derivative may be produced.
  • a preferred tyrosine kinase derivative includes the tyrosine kinase domain linked to the C-terminal domain.
  • the two domains may be produced as separate molecules, and mixed together to achieve tyrosine phosphorylation of the C-terminus-derived polypeptide,
  • the probe comprising a tyrosine-phosphorylated C-terminal portion of the tyrosine kinase, as described herein can be produced by recombinant means in the form of a fusion protein.
  • a "fusion protein” may refer to a fused protein comprising a bacterial protein and a polypeptide of interest such as a protein having an SH2 domain.
  • a fusion protein may also be an artificially constructed tyrosine kinase-like derivative, wherein a DNA sequence encoding the tyrosine kinase domain has been linked to a selective enzymatic cleavage site, which, in turn, is linked to a tyrosine kinase C-terminal domain having one or more tyrosine residues which can be phosphorylated by the kinase.
  • Such a genetic construct encoding this type of "fusion protein” can be inserted into an expression vehicle and expressed in a bacterial or eukaryotic host. Once expressed, such a fusion protein can be allowed to autophosphorylate, wherein the kinase acts to phosphorylate the tyrosine residues in the C-terminal domain. Following this phosphorylation, use of the appropriate enzyme will cleave at the selective cleavage site, thus separating the N-terminal kinase from the C-terminal phosphorylated polypeptide, which can now serve as a probe.
  • selective cleavage site refers to an amlno acid residue or residues which can be selectively cleaved with either chemicals or enzymes and where cleavage can be achieved in a predictable manner.
  • a selective enzymatic cleavage site is an amino acid or a peptide sequence which is recognized and hydrolyzed by a proteolytic enzyme. Examples of such sites include trypsin or chymotrypsin cleavage sites.
  • the selective cleavage site is comprised of the sequence ile-Glu-Gly-Arg (SEQ ID NO: 15), which is recognized and cleaved by blood coagulation factor Xa.
  • the selective cleavage site has the sequence Leu-Val-Pro-Arg (SEQ ID NO:16), which is recognized and cleaved by thrombin.
  • an oligonucleotide sequence 5' to the sequence coding for the enzyme recognition site can be included, and may vary in length.
  • 13 nucleotides are situated between the codon for Ils (the start of the factor Xa recognition site) and the 3' end of the sequence encoding the tyrosine kinase domain.
  • the ile-Glu-Gly-Arg (SEQ ID NO:15) sequence is introduced between. the tyrosine kinase domain and the Co terminal domain.
  • the Leu-Val-Pro-Arg (SEQ ID NO:16) sequence is introduced.
  • the proteins having this cleavage site are expressed in bacteria using standard methods. Thereafter, autophosphorylation of the C-terminal domain, preferably with ( ⁇ 32 P) adenosine triphosphate, is allowed to occur, followed by selective cleavage of the tyrosine-phosphorylated C-terminal domain with the appropriate cleaving agent, e.g., factor Xa.
  • the appropriate cleaving agent e.g., factor Xa.
  • the present invention also provides a method for mapping a gene, preferably a human gene, which encodes a target protein for a tyrosine kinase (such as a GRB protein as defined herein), to a particular human chromosome.
  • This method combines the new expression cloning method described herein with one of several known techniques for mapping a gene to a particular chromosome.
  • a clone such as a lambda gtll clone, containing a DNA insert encoding a GRB protein, is identified using the expression cloning methods disclosed herein.
  • the insert may be further subcloned, if desired, using methods well-known in the art, and a probe constructed, either by direct labeling of the nucleic acid of the clone or by producing an oligonucleotide probe corresponding to a unique. portion of the clone's sequence (see: Sambrook, J. et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); and Ausubel, supra).
  • This labeled probe can is then used in a hybridization assay with commercially available blots, such Chromosome Blots from Bios Corporation (New Haven, Conn.) which contain DNA from a panel of human-hamster somatic cell hybrids (Kouri, R. E. et al., Cytoqenet. Cell Genet. 51:1025 (1989)).
  • blots such Chromosome Blots from Bios Corporation (New Haven, Conn.) which contain DNA from a panel of human-hamster somatic cell hybrids (Kouri, R. E. et al., Cytoqenet. Cell Genet. 51:1025 (1989)).
  • the gene is mapped to a particular human chromosome. In this way, linkage is established to known human genes (or diseases caused by mutations therein) present on this chromosome.
  • the GRB gene can be mapped more precisely to other human genes.
  • the tyrosine-phosphorylated tyrosine kinase C-terminal probe polypeptide of the present invention are useful in methods for screening drugs and other agents which are capable of modulating cell growth control that occurs via signal transduction through tyrosine kinases.
  • an affinity probe is created which can be used to isolate and purify molecules from complex mixtures which are capable of binding to the affinity probe.
  • an affinity probe is useful for detecting the presence in a biological fluid of a molecule capable of binding the tyrosine-phosphorylated probe or the GRB protein.
  • chemical agents can be tested for their capacity to interact with the probe or GRB.
  • tyrosine phosphorylation is linked to cell growth and to oncogenic transformation. Disruption of the action of a GRB in the cell may prevent or inhibit growth, and might serve as means to counteract development of a tumor. Furthermore, a mutation in the C-terminal portion of the tyrosine kinase or the GRB, or a disregulation in their mutual interactions, may promote susceptibility to cancer.
  • the insulin receptor is also a receptor tyrosine kinase, and tyrosine phosphorylation in cells bearing InsR is associated with normal physiological function.
  • InsR The insulin receptor
  • tyrosine phosphorylation in cells bearing InsR is associated with normal physiological function.
  • Subnormal levels or activity of a GRB protein may act to remove a normal counterregulatory mechanisms. It is expected that overexpression or overactivity of a GRB protein could inhibit or totally prevent the action of insulin on cells, leading to diabetes (of an insulin-resistant variety). Thus susceptibility to diabetes may be associated with GRB protein dysregulation.
  • methods of the present invention for identifying normal or mutant GRB protein genes, or for detecting the presence or the amount of GRB protein in a cell can serve as methods for identifying susceptibility to cancer, diabetes, or other diseases associated with alterations in cellular metabolism mediated by tyrosine kinase pathways.
  • the present invention provides methods for evaluating the presence, and the level of normal or mutant GRB protein in a subject. Altered expression of these proteins, or presence of a mutant GRB protein, in an individual may serve as an important predictor of susceptibility to oncogenic transformation and the development of cancer. Alternatively, altered expression of GRB protein may serve as an important predictor of susceptibility to diabetes.
  • Oligonucleotide probes encoding various portions of the GRB protein are used to test cells from a subject for the presence DNA or RNA sequences encoding the GRB protein.
  • a preferred probe would be one directed to the nucleic acid sequence encoding at least 4 amino acid residues, and preferably at least 5 amino acid residues of the GRB-1, GRB-2, GRB-3, GRB-4 GRB-7 or GRB-10, protein of the present invention, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids.
  • Qualitative or quantitative assays can be performed using such probes. For example, Northern analysis (see Example III, below) is used to measure expression of an GRB protein mRNA in a cell or tissue preparation.
  • Such methods can be used even with very small amounts of DNA obtained from an individual, following use of selective amplification techniques.
  • Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have long been recognized- Typically, such methodologies involve the introduction of the nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al. (U.S. Pat. No. 4,237,224), Sambrook et al. (supra), Ausubel et al, supra, etc.
  • PCR polymerase chain reaction
  • the invention is directed to target proteins of eukaryotic tyrosine kinases, which include, as non-limiting examples, GRB proteins such as GRB-1, GRB-2, GRB-3, GRB-4 GRB-7 or GRB-10 proteins are included.
  • GRB proteins such as GRB-1, GRB-2, GRB-3, GRB-4 GRB-7 or GRB-10 proteins are included.
  • the invention is directed to recombinant eukaryotic GRB proteins.
  • the invention provides the naturally occurring protein molecule substantially free of other proteins with which it is natively associated. "Substantially free of other proteins or glycoproteins" indicates that the protein has been purified away from at least 90 per cent (on a weight basis), and from even at least 99 per cent if desired, of other proteins and glycoproteins with which it is natively associated, and is therefore substantially free of them.
  • GRB-1, GRB-2, GRB-3, GRB-4 GRB-7 or GRB-10 protein can be achieved by subjecting the cells, tissue or fluids containing the GRB-1, GRB-2, GRB-3, GRB-4 GRB-7 or GRB-10 protein to standard protein purification techniques such as immunoadsorbent columns bearing monoclonal antibodies reactive against the protein.
  • GRB-1, GRB-2, GRB-3, GRB-4 GRB-7 or GRB-10, or other eukaryotic GRB protein can be isolated and purified using as an affinity probe, the probe of the present invention which is a tyrosine-phosphorylated C-terminal domain of a tyrosine kinase, or a functional derivative thereof.
  • the purification can be achieved by a combination of standard methods, such as ammonium sulfate precipitation, molecular sieve chromatography, and ion exchange chromatography.
  • GRB-1 proteins of the present invention can be biochemically purified from a variety of cell or tissue sources.
  • tissues such as mammalian placenta or brain are preferred.
  • the invention is also directed to a recombinant nucleic acid molecule having a nucleotide sequence that encodes at least one of the GRB proteins of the invention, including, but not limited to GRB-1, GRB-2, GRB-3, GRB-4, GRB-7, or GRB-10 proteins. Given their potential role in signal transduction, such GRB proteins may be referred to herein as "adaptor proteins" Further, the invention is directed to a recombinant nucleic acid molecule having a nucleotide sequence that selectively hybridizes to the complement of the recombinant nucleic acids which encode GRB proteins, as described above.
  • Nucleic acids may refer, for example, to cDNA or to genomic DNA.
  • the recombinant nucleic acids described above may be contained within a recombinant vector, such as an expression vector containing a recombinant nucleic acid having a nucleotide sequence as described above, operatively associated with an element that controls expression of the nucleotide sequence in a host cell.
  • Selective hybridization refers to nucleic acid hybridization under standard stringency conditions, which are well known to those of skill in the art. (See, for example, Sambrook, supra, and Ausubel, supra.)
  • the recombinant nucleic acids described above may also be contained within an engineered host cell, which may be of either eukaryotic or prokaryotic origin. Such an engineered host cell may further contain an element that controls the expression, in the host cell, of the nucleotide sequence of the above-described recombinant nucleic acids. Such an engineered host cell may be of prokaryotic or eukaryotic origin.
  • the polypeptide can be synthesized substantially free of other proteins or glycoproteins of mammalian origin in a prokaryotic organism or in a non-mammalian eukaryotic organism, if desired.
  • a recombinant GRB-1, GRB-2, GRB-3, GRB-4 GRB-7 or GRB-10 molecule produced in mammalian cells, such as transfected COS, NIH-3T3, or CHO cells for example, is either a naturally occurring protein sequence or a functional derivative thereof. Where a naturally occurring protein or glycoprotein is produced by recombinant means, it is provided substantially free of the other proteins and glycoproteins with which it is natively associated.
  • the tyrosine-phosphorylated C-terminal domain probe of the present invention can be synthesized using a peptide synthesis method wherein phosphotyrosine is provided in place of tyrosine, resulting in direct synthesis of the phosphorylated form of the polypeptide. See, e.g., Staerkaer et al, Tetrahedron Letters 32:5289-5392 (1991); Shoelson et al Tetrahedron Letters 32:6061 (1991), which references are entirely incorporated herein by reference).
  • the present invention also provides "functional derivatives" of the tyrosine-phosphorylated C-terminal domain polypeptide and or the GRB-1, GRB-2, GRB-3, GRB-4 GRB-7 or GRB-10 proteins.
  • мно By “functional derivative” is meant a “fragment,” “variant,” “analog,” or “chemical derivative” of the GRB protein, which terms are defined below.
  • a functional derivative retains at least a portion of the function of the native protein which permits its utility in accordance with the present invention.
  • a "fragment" of any of the proteins or polypeptides of the present invention refers to any subset of the molecule, that is, a shorter peptide.
  • variant of the protein refers to a molecule substantially similar to either the entire peptide or a fragment thereof.
  • Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide, using methods well- known in the art.
  • substantially corresponding to the amino acid sequence of in the context of the present refers to a protein containing conservative amino acid substitutions, known in the art and as described herein, that would be expected to maintain the functional biological activity of the referenced sequence, and/or target protein binding characteristics.
  • substitutions can be readily determined without undue experimentation by using known conservative substitutions, as known in the art.
  • known software can be used to provide such conservative substitutions according to the present invention.
  • the program "BESTFIT" can be used to provide conservative amino acid substitutions of a define sequence, e.g., defined as having a score of ⁇ 0.4, 0.6, 0.8 or 1.0 depending on the type of protein used. See e.g., Gribskov and Burgess, Nucl. Acid. Res. 14:6745 (1984), which is entirely incorporated by reference.
  • Variant peptides may be conveniently prepared by direct chemical synthesis of the variant peptide using methods well- known in the art.
  • amino acid sequence variants of the peptide can be prepared by mutations in the DNA which encodes the synthesized peptide.
  • Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity. Mutations that will be made in the DNA encoding the variant peptide must not alter the reading frame and preferably will not create complementary regions that could produce secondary mRNA structure (see European Patent Publication No. EP 75,444).
  • these variants ordinarily are prepared by site-directed mutagenesis (as exemplified by Adelman et al., DNA 2:183 (1983)) of nucleotides in the DNA encoding the peptide molecule, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture (see below).
  • the variants typically exhibit the same qualitative biological activity as the nonvariant peptide.
  • Amino acid substitutions in the context of the present invention include substitutions wherein at least one amino acid residue in the peptide molecule, and preferably, only one, has been removed and a different residue inserted in its place.
  • amino acid sequences substantially corresponding to a given sequence can be made without undue experimentation and then routinely screened for tyrosine kinase binding activity using known methods or those disclosed herein, such that one of ordinary skill in the art can determine which substitutions provide tyrosine kinase target proteins according to the present invention. For example, once target protein sequences are determined, such as for GRB-1, GRB-2, GRB-3, GRB-4 GRB-7 or GRB-10, conservative amino acid substitutions can be made to provide target proteins having amino acid sequences which substantially correspond to the determined target protein sequences.
  • the preferred bacterial host for this invention is E. coli. In other embodiments, other bacterial species can be used. In yet other embodiments, eukaryotic cells may be utilized, such as, for example, yeast, filamentous fungi, or the like. Use of these cell types are well known in the art. Any host may be used to express the protein which is compatible with replicon and control sequences in the expression plasmid. In general, vectors containing replicon and control sequences are derived from species compatible with a host cell are used in connection with the host. The vector ordinarily carries a replicon site, as well as specific genes which are capable of providing phenotypic selection in infected or in transformed cells.
  • the expression of the fusion protein can also be placed under control with other regulatory sequences which may be homologous to the organism in its untransformed state.
  • Preferred promoters can include a T7 promoter.
  • Such preferred promoters express the human gene as a fusion protein such as the T7 capsid protein P10 under control of the T7 promoter.
  • Such expression systems are commercially available, as the ⁇ EXlox vector from Novagen, Inc. (Madison, Wis.).
  • the recombinant T7 vector containing a human gene, encoding such proteins obtainable by methods of the present invention such as GRB-1, GRB-2, GRB-3, GRB-4 and GRB-7, as, e.g., a T10 fusion protein.
  • the recombinant T7 vector can then be used to transform a bacteria, such as E. coli, by infection with a phage containing the recombinant T7 vector under lac control, such lacUV5 control.
  • a bacteria such as E. coli
  • lac control such lacUV5 control.
  • IPTG Induction of the infected, successfully transformed bacteria or other suitable host cell, by IPTG generates the T7 polymerase which then initiates transcription of the fusion protein encodedby the phage library.
  • T7 polymerase provide human gene library plaques that have stronger signals than obtained by the use of bacterial RNA polymerases, such as E. coli RNA polymerase.
  • the use of a T7 polymerase expression system is particularly suitable for library screening when there as thousands of small plaques per plate.
  • T7 expression system The major advantage of the use of a T7 expression system is the high level of protein expression due to the greater activity of the T7 polymerase versus E. coli tNA polymerase, and because fusion proteins using the smaller phage fusion protein gene, such as the T10 gene fragment (26 kd versus the 110 kd B-galactosidase of ⁇ gt11 expression library) yields more stable expression and that its hydrophobic character promotes binding to nitrocellulose.
  • T7 phages In addition to directional cloning, the use of T7 phages also allow for automatic conversion to a PET plasmid (see, e.g., Palazzalo et al., Gene 88, 25 (1990)) which can be useful for expression of a fusion protein for antibody production.
  • This invention is also directed to an antibody specific for an epitope of the GRB-1, GRB-2, GRB-3, GRB-4 GRB-7 or GRB-10 protein and the use of such an antibody to detect the presence of, or measure the quantity or concentration of, the GRB protein in a cell, a cell or tissue extract, or a biological fluid.
  • antibody is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, and anti-idiotypic (anti-Id) antibodies.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.
  • Monoclonal antibodies are a substantially homogeneous population of antibodies to specific antigens.
  • MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature 256:495-497 (1975) and U.S. Pat. No. 4,376,110.
  • Such antibodies may be of any immunoglobulin class including igG, IgM, IgE, IgA, GILD and any subclass thereof.
  • the hybridoma producing the mAbs of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo production makes this the presently preferred method of production.
  • MAbs of isotype may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.
  • Chimeric antibodies are molecules different portions of which are derived from different animal species , such as those having variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies and methods for their production are known in the art (Cabilly et al, Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984); Boulianne et al., Nature 312:643-646 (1984); Cabilly et al., European Patent Application 125023 (published Nov.
  • An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
  • An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., mouse strain) as the source of the mAb with the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody).
  • the anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
  • the anti-anti-Id may be epitopically identical to the original mAb which induced the anti-Id.
  • antibodies to the idiotypic determinants of a mAb it is possible to identify other clones expressing antibodies of identical specificity.
  • mAbs generated against the GRB protein of the present invention may be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice.
  • Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-IdmAbs.
  • the anti-IdmAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional BALB/c mice.
  • KLH keyhole limpet hemocyanin
  • Sera from these mice will contain anti-anti-id antibodies that have the binding properties of the original mAb specific for a GRB protein epitope.
  • the anti-Id mAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as GRB protein- ⁇ .
  • antibody is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab') 2 , which are capable of binding antigen.
  • Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).
  • Fab and F(ab') 2 and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of GRB protein according to the methods disclosed herein for intact antibody molecules.
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab') 2 fragments).
  • an antibody is said to be “capable of binding” a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • epitope is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody.
  • Epitopes or "antigenic determinants” usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • an "antigen” is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen.
  • An antigen may have one, or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens.
  • the antibodies, or fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect the presence of cells which express the GRB protein. This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorometric detection.
  • the antibodies (of fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of GRB proteins.
  • In situ detection may be accomplished by removing a histological specimen form a patient, and providing the a labeled antibody of the present invention to such a specimen.
  • the antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample.
  • Such assays for GRB protein typically comprises incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leukocytes, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capable of identifying GRB protein, and detecting the antibody by any of a number of techniques well-known in the art.
  • a biological sample such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leukocytes, or cells which have been incubated in tissue culture
  • the biological sample may be treated with a solid phase support or carrier such as nitrocellulose, or other solid support or carrier which is capable of immobilizing cells, cell particles or soluble proteins.
  • a solid phase support or carrier such as nitrocellulose, or other solid support or carrier which is capable of immobilizing cells, cell particles or soluble proteins.
  • the support or carrier may then be washed with suitable buffers followed by treatment with the detectably labeled GRB protein-specific antibody.
  • the solid phase support or carrier may then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on said solid support or carrier may then be detected by conventional means.
  • solid phase support By “solid phase support”, “solid phase carrier”, “solid support”, “solid carrier”, “support” or “carrier” is intended any support or carrier capable of binding antigen or antibodies.
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support or carrrier configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports or carriers include polystyrene beads. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • binding activity of a given lot of anti-GRB-1, anti-GRB-2, anti-GRB-3, Anti-GRB-4 or anti-GRB-7, antibody may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • GRB-specific antibody can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA).
  • EIA enzyme immunoassay
  • This enzyme when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means.
  • Enzymes which can be used detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6- phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection may be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay RIA
  • the radioactive isotope can be detected by such means as the use of a ⁇ counter or a scintillation counter or by autoradiography.
  • fluorescent labelling compounds fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the antibody can also be detectably labeled using fluorescence emitting metals such as 152 EU, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (EDTA).
  • fluorescence emitting metals such as 152 EU, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (EDTA).
  • the antibody also can be detectably labeledby coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • the antibody molecules of the present invention may be adapted for utilization in a immunometric assay, also known as a "two-site” or “sandwich” assay.
  • a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support. or carrier and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
  • Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen form the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support or carrier is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support or carrier through the unlabeled antibody, the solid support or carrier is washed a second time to remove the unreacted labeled antibody.
  • a simultaneous assay involves a single incubation.step as the antibody bound to the solid support or carrier and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support or carrier is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support or carrier is then determined as it would be in a conventional "forward" sandwich assay.
  • stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support or carrier after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support or carrier is then determined as in the "simultaneous" and "forward" assays.
  • the intracellular portion of the EGFR which includes the tyrosine kinase domain and the carboxy terminal domain, was purified from recombinant baculovirus which expressed cDNA complementary to the intracellular domain of the human EGFR, as described previously (Hsu, C-Y. et al., Cell Growth and Differentiation 1:191-200 (1990)).
  • the recombinant protein (2 ⁇ g) was then phosphorylated with ( ⁇ - 32 P)ATP (200 ⁇ Ci, 6000 Ci/Mmol)., at 4° C.
  • the concentrated protein was then digested with cyanogen bromide (CNBr) in 70% formic acid for 14 hours at room temperature (see also Example VI, below). Samples were then washed three times with water, dried and resuspended in binding buffer to a concentration of 2 ⁇ 10 6 cpm/ml.
  • CNBr cyanogen bromide
  • TrpE and TrpE/GAP-SH2 were obtained from the laboratory of Dr. Tony Pawson and/or prepared as previously described (Moran, M. F. et al., Proc. Natl. Acad. Sci. USA 87:8622-8626 (1990)). Filter binding studies were performed according to published methods (Schneider, W. J. et al., Proc. Natl. Acad. Sci. 76:5577-5581 (1979); Daniel, T. O. et al., J. Biol. Chem. 258:4606-4611 (1983)) with minor modifications. Various concentrations of either bacterially expressed TrpE fusion protein or bacterial protein alone were spotted onto nitrocellulose filters.
  • the above method permitted detection of specific binding of the EGFR C-terminal domain to less than 5 ng of a bacterially expressed GAP-SH2 fusion protein.
  • the binding was specific, since it required tyrosine phosphorylation of the probe and did not occur when irrelevant proteins were applied to nitrocellulose filters.
  • the tyrosine phosphorylated C-terminal tail of the EGFR was used as a probe to screen expression libraries from several different human tissues as described above.
  • the approach to screening is outlined in FIG. 2. Numerous positive clones have been identified so far using this approach, of which two have been analyzed in detail.
  • lambda gtll phage were plated at a density sufficient to produce 4 ⁇ 10 4 plaques per 150 mm agar plate. A total of six plates were initially screened. After incubation of the plates for 4 hours at 42° C., the plates were overlaid with nitrocellulose filters which had been impregnated with isopropyl-B-D-thiogalactopyranoside (IPTG), as previously described (MacGregor, P. F. et al., Oncogene 5:451-458 (1990)). Incubation was continued overnight at 37° C.
  • IPTG isopropyl-B-D-thiogalactopyranoside
  • the filters were then removed, washed with tBST (10 mM Tris-HCl, pH8, 150 mMNaC1, and 0.05% triton X-100) at room temperature, and then blocked in EBB (20 mM HEPES, pH 7.5, 5 mM Mg/Cl, 1 mM KCl) buffer containing 5% carnation dry milk for 1 hour at 4° C., as described (MacGregor et al., supra). Following blocking, labelled tyrosine phosphorylated carboxy-terminus (C-terminus) probe was added at a concentration of 1.6 ⁇ 10 -4 ⁇ g/ml, and incubation was continued overnight. The filters were then washed 3 times at room temperature in PBS containing 0.2% Triton X-100. Filters were dried and exposed to Kodak XAR-5 film at -80° C.
  • Agar plugs corresponding to the positive clones, were collect from the plates and placed in 1 ml of SM media. After allowing the phages to diffuse from the agar, the phages were replated and rescreened as described above. Those phages that demonstrated enrichment on subsequent screening were isolated and sequence. Lambda gtll phage DNA was isolated by the plate lysate method according to Maniatis et al., and subcloned into EcoRI-digested M13 MP19 (Maniatis et al., 1982). Single stranded DNA was isolated and sequenced by the dideoxy chain termination method using the Sequenase DNA sequencing kit (United States Biochemical).
  • FIG. 3A A single plaque, clone ki4 (FIG. 3A) was isolated. On subsequent screening this clone demonstrated enrichment, and on tertiary screening all plaques bound the probe (FIG. 3B).
  • Clone ki4 contained an insert of about 900 nucleotides, which, upon induction of the lac promoter with IPTG, produced a fusion protein which could bind the EGFR.
  • the size of the fusion protein predicted that the cDNA insert coded for a protein of about 300 amino acids, which was the size expected if the cDNA contained a single large open reading frame.
  • the library was replated as described above. After incubation of the plates for 8 hours at 37° C., the plates were cooled for 1 hour at 4° C. following which the phage DNA was transferred to nitrocellulose filters. The filters were denatured in a solution of 0.2N NaOH and 1.5 M NaCl and then baked in vacuo for 2 hours at 80° C. (Sambrook, J. et al., (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). After prehybridization of the filters for 1 hour at 42° C., 32P-labelled DNA probe was added and hybridization was continued overnight at 42° C.
  • GRB-1 Protein Contains SH2 and SH3 domains
  • GRB-1 protein sequence by comparison to sequences in the Genbank database revealed the presence of two stretches of about 100 amino acids, starting at amino acids 333 and 624, with sequence homology to SH2 domains of other proteins known to interact with the EGFR (FIG. 5). While GRB-1 displayed striking homology to other SH2 domains at the protein level, it revealed no significant homology at the DNA level. GRB-1 also contained a segment of about 50 amino acids, located in the N-terminal region, which had sequence homology to SH3 domains (FIG. 4A to 4I and 5).
  • GRB-1 is similar to some other substrates which have been found to interact with the EGFR, such as PLC- ⁇ and GAP, in that GRB-1 contains two SH2 domains and a single SH3 domain. However, unlike these substrates, GRB-1 contains no homology to any known catalytic domain,. and in this regard resembles the protein encoded by the arian sarcoma virus, v-crk.
  • GRB-1 lacked a consensus ATP-binding domain, and did nod display sequence homology with any serine/threonine kinase or tyrosine kinase.
  • the SH2 domain is thought to provide a common motif by which enzymatically distinct signalling molecules can be coupled to activated receptors with tyrosine kinase activity (Moran, M. F. et al., Proc. Natl. Acad. Sci. USA 87:8622-8626 (1990); Anderson, D. et al., Science 250:979-982 (1990)).
  • GRB-1 In addition to containing two SH2 domains, GRB-1 also contains an SH3 domain.
  • the SH3 domain is a non-catalytic domain of about 50 amino acid residues which is shared among many SH2-containing proteins. Since SH3 domains are also found in cytoskeletal proteins, such as spectrin and fodrin, the function of this domain could be to localize these proteins to the membrane or submembrane cytoskeleton where they would interact with other molecules.
  • GRB-1 The gene v-crk encodes a protein which is composed primarily of a viral gag protein fused to an SH2 and SH3 domain (Mayer, B. J. et al., Nature 332:272-275 (1988)). Both GRB-1 and the p47 gag-crk protein have no homology with any known catalytic domains. However, chicken embryo fibroblasts transformed with p47 gag-crk display elevated levels of phosphotyrosine-containing proteins (Mayer, B. J. et al., supra; Proc. Natl. Acad. Sci. USA 87:2638-2642 (1990); Matsuda, M. et al., Science 248:1537-1539 (1990)) .
  • v-crk product Since the v-crk product has been shown to bind several phosphotyrosine-containing proteins in v-crk transformed cells, it may be that the function of c-crk is to act as a bridge between kinases and substrates.
  • GRB-1 like GAP and PLC-7, contains two SH2 domains, the combination of which may be ideally suited for linking other proteins to activated tyrosine kinase molecules.
  • RNA Total cellular RNA was prepared from monkey tissue by the guanidinium isothiocyanate/cesium chloride method described by Sambrook, J. et al., (supra).
  • Poly (A)+RNA was prepared by oligo(dT) cellulose chromatography.
  • RNA was size fractionated by electrophoresis in a 1.2% agarose/2.2M formaldehyde gel, transferred onto a nylon membrane by capillary action and baked at 80° C. for 2 hours. Following prehybridization, the blot was hybridized with a (32P)-nick-translated DNA probe which was prepared as descried above. Hybridization was carried out overnight at 42° C.
  • the 3.6, 6.6 and 7.0 kb transcripts may represent alternatively spliced forms of mRNA, or may encode for distinct but related mRNA species.
  • Polyclonal antibodies were produced by immunizing rabbits with the S-galactosidase fusion protein expressed by the initial isolated phage clone, ki4.
  • E. coli GAG 456 bacteria obtained from Dr. Michael Snyder, Yale University
  • recombinant phage ki4 at a multiplicity-of-infection of 10
  • g-galactosidase fusion protein was recovered from the protein pellet after 1.5 hours.
  • Protein extracts were prepared, separated on a 6% SDS-Sel, and the band corresponding to the fusion protein excised from gel and used for immunization.
  • Human glioblastoma cell line U1242, rat bladder carcinoma cell line NBT II, and NIH3T3 cells were grown to confluence in DMEM medium supplemented with 10% fetal bovine serum. Cells were labelled with ( 35 S)-methionine (50 ⁇ Ci/ml) in 0.5% fetal bovine serum and lysed after 12 hours as previously described (Marg01is, B. et al., Cell 57:1101-1107 (1989)).
  • the beads were washed three times with a solution containing 20 mM HEPES, pH 7.5, 300 mM NaCl, 10% glycerol, 1% Triton X-100, 0.1% SDS, and 1% sodium deoxycholate. After boiling in sample buffer proteins were separated on a 8% SDS-gel.
  • GRB-1 encodes for a protein with an expected molecular weight of 85 kDa
  • p85 phosphatidylinositol
  • MTAg middle T-antigen
  • GRB-1 is most likely not a phospholipid kinase.
  • GRB-1 exhibits 97% sequence identity with murine and bovine p85.
  • GRB-1 is the human counterpart of p85.
  • Recombinant p85 is able to bind to the activated PDGFR or EGFR, but does not itself contain intrinsic PI3 kinase activity.
  • p85 is found associated with a 110 kDa tyrosine phosphorylated protein which may be the catalytic subunit of the PI3 Kinase. While the exact relationship between PI3 kinase and p85 is not known, overexpression of p85 modulates the interaction between PI3 kinase and the PDGFR. p85 could function as a regulatory subunit or as a bridge between activated receptors and the PI3 kinase.
  • the Tyrosine Phosphorylated Carboxy-terminus of the EGF Receptor is a Binding Site for GAP and PLC- ⁇
  • the cell lines CD126 (Margolis, B. L. et al., J. Biol. Chem. 264:10667-10671 (1989a), HER14, K721 (Honegger, A. M. et al., Cell 51:199-209 (1987); Honegger, A. M. et al., Mol. Cell. Biol. 7:4567-4571 (1987)) were used as sources for wild-type EGF receptor, kinase-negative (kin - ) EGF receptor and C-terminal (C-terminal) truncated EGF receptor, respectively.
  • the intracellular domain of the EGF receptor (EGFR-C) was purified from a baculovirus expression system (Hsu, C-. J.
  • trpE fusion proteins containing the GAP SH2 domain (GAP residues 171-448, FIG. 9B) has been described by Moran, M. F. et al., Proc. Natl. Acad. Sci. USA 87: 8622-8626 (1990).
  • Bacterial lysates containing trpE/GAP SH2 fusion proteins were prepared by resuspending 1 g of bacteria in 3 ml of 50 mM Tris pH 7.5, 0.5 mM EDTA, 0.1 mM PMSF. After incubation at 4° C.
  • Bacterial lysates were diluted 1:100 in the 1% Triton lysis buffer with proteinase and phosphatase inhibitors as described above and were precleared with protein A-Sepharose.
  • FIG. 9A The following anti-EGFR antibodies (FIG. 9A) were used: (a) mAb108, a monoclonal antibody directed against domain III of the extracellular domain (Lax, I. et al., EMBO J. 8: 421-427 (1989)); (b) antipeptide antibody RK2 specific for residues 984-996; (c) antipeptide antibody C specific for residues 1176-1186; and (d) antipeptide antibody F, specific for residues 56-676.
  • mAb108 a monoclonal antibody directed against domain III of the extracellular domain
  • RK2 antipeptide antibody RK2 specific for residues 984-996
  • antipeptide antibody C specific for residues 1176-1186
  • antipeptide antibody F specific for residues 56-676.
  • trpE fusion proteins a mouse monoclonal antibody against trpE (Oncogene Science) bound to agarose linked anti-mouse IgG (Sigma) was utilized.
  • Unstimulated cells were grown to confluence in Dulbecco's Modified Eagle Medium with 10% calf serum and starved overnight in 1% fetal calf serum prior to lysis in a 1% Triton X-100 lysis buffer containing proteinase and phosphatase inhibitors. EGF receptors were immunoprecipitated utilizing antibodies bound to protein A-Sepharose.
  • HNTG 20 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Triton X-100 and 10% glycerol
  • HNTG 20 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Triton X-100 and 10% glycerol
  • autophosphorylation was induced by the addition of 5 mM MnCl 2 and 30 ⁇ M ATP.
  • Controls were incubated with Mn 2+ only.
  • lysate containing either PLC- ⁇ (from 3TP1 cells) or the bacterial fusion proteins was added. After allowing binding to proceed for 90 min, three further washes with HNTG were performed and samples were run on an SDS gel and immunoblotted.
  • EGFR-C was phosphorylated at 4° C. with MnCl 2 and ATP sometimes in the presence of ( ⁇ - 32 P)ATP (NEN/Dupont, 6000 Ci/mmol).
  • the receptor preparation was then resuspended in 20 mM HEPES, pH 7.5, with 100 ⁇ gBSA and concentrated in a Centricoh 10 (Amicon) to 50 ⁇ l. Then 240 ⁇ l 88% formic acid was added with two grains of CNBr and the samples were stored under nitrogen in the dark for 14 h at room temperature. Samples were dried and washed three times with water in a Speed-Vac (Savant) and then resuspended in 1% Triton lysis buffer.
  • Savant Speed-Vac
  • FIG. 9A A comparison was performed of the binding of PLC- ⁇ to wild-type and mutant EGFRs (FIG. 9A).
  • wild-type and mutant receptors from transfected NIH-3T3 cells were immunoprecipitated and some of the receptor immunoprecipitates were allowed to undergo in vitro autophosphorylation with ATP and Mn 2+ (Margolis, B. et al., Mol. Cell. Biol. 10:435-441 (1990a)).
  • lysates from NIH-3T3 cells which overexpress PLC- ⁇ (Margolis, B. et al., Science 248: 607-610 (1990b)) were added and binding allowed to proceed for 90 min. at 4° C.
  • FIG. 10A-10B also demonstrates that PLC- ⁇ cannot bind to the kin - mutant receptor.
  • the kin - receptor was cross-phosphorylated with the CD126 receptor (Honegger, A. M. et al., Proc. Natl. Acad. Sci. USA 86: 925-929 (1989)). This resulted in normalization of PLC- ⁇ binding to wild-type levels. This suggested that phosphorylation of the kin - receptor was sufficient to normalize binding to PLC- ⁇ .
  • this receptor was cross-phosphorylated with a soluble, baculovirus-expressed EGFR cytoplasmic domain (EGFR-C) that does not bind to the mAb 108 (FIG. 9A).
  • EGFR-C soluble, baculovirus-expressed EGFR cytoplasmic domain
  • the trpE/GAP SH2 fusion protein bound with a higher stoichiometry to full length EGFR than did PLC- ⁇ .
  • the fusion protein was not tyrosine phosphorylated by the EGFR.
  • the trpE/GAP SH2 protein much better to the phosphorylated full length receptor compared to the CD126 deletion mutant (FIG. 13A).
  • FIG. 13B cross-phosphorylation of the kiff full length EGF receptor by the EGFR-C allowed it to bind the trpE/GAP SH2 protein.
  • the poor binding to the CD126 deletion mutant suggested that at least part of the binding site for the molecule was in the C-terminus. Yet an effect, possibly allosteric, of this deletion on the overall conformation of the receptor could not be excluded. Therefore, the binding of PLC- ⁇ and trpE/GAP SH2 to a C-terminal fragment of the EGFR was examined. In the EGFR, the most C-terminal methionine residue is found at position 983; CNBr cleavage therefore generates a 203 amino acid fragment which contains all the known autophosphorylation sites. This protein fragment is recognized by an antibody specific for the EGFR C-terminus, anti-C (FIG. 9A).
  • EGFR-C was autophosphorylated with ( ⁇ - 32 P)ATP and a 32 P-labeled CNBr C-terminal fragment was generated. As shown in FIG. 15, this fragment bound to the trpE/GAP SH2 fusion protein but not to trpE. In total, these findings demonstrate that direct binding to the tyrosine phosphorylated C-terminus contributes at least in part to the specific binding of SH2 and SH3 domain proteins to the EGFR.
  • the phosphotyrosine residues either comprise a part of the binding site or locally alter the conformation of this region, allowing binding. It is unlikely that phosphotyrosine alone constitutes the binding site. For example, phosphotyrosine alone cannot interfere with the binding of P47 gag-crk to phosphotyrosine-containing proteins (Matsuda et al., supra). Additionally, PLC- ⁇ does not bind to activated all molecules that contain phosphotyrosine residues, such as the CSF-1 receptor (Downing, J. R. et al., EMBO J. 8:3345-3350 (1989)). Similarly, the binding of PLC- ⁇ to PDGFR does not appear to be identical to GAP binding; different SH2 and SH3 domain-containing proteins may have different binding specificities (Kazlauskas et al., supra).
  • RESULTS The intracellular domain of the EGFR, which includes the tyrosine kinase and carboxy terminal domain, was purified from a recombinant baculovirus expression system as described (Margolis Mol. Cell. Biol. 10:435-441 (1990) and EMBO J. 9:4375-4390 (1990); Skolnik et al. Cell 65:83-90 (1991).
  • the recombinant protein was phosphorylated with ( 32 P) ⁇ -ATP, washed, and cyanogen bromide digested to yield a 204 residue carboxyterminal tail containing all five phosphorylated tyrosine residues (Margolis Mol. Cell. Biol.
  • oligo (dT) ⁇ gt11 constructed from mRNA isolated from human brain stem, was obtained from M. Jaye (Rhone Poulenic-Rorer Pharmaceuticals) and is readily available from commercial sources. Screening of the library was performed as previously described (Skolnik et al. Cell 65:83-90 (1991)). cDNA inserts isolated from positive recombinant phage that bound the EGFR were subcloned into M13 and sequenced by the dideoxy chain termination method, using the Sequenase 2.0 kit (U.S.B). Since the initial clone isolated by expression/cloning did not contain the 5' ends of the gene, the library was rescreened, using the clone 2-4 insert as a DNA probe.
  • HER14 are NIH 3T3 cells (clone 2.2) which express approximately 400,000 wild type human EGF receptors per cell (Honeggar et al. Cell 51:199-209 (1987)). HER14 cells were maintained in Dulbecco's modified Eagles medium (DMEM) containing 10% calf serum (CS). Prior to stimulation, cells were cultured for 18 hours in DMEM/1% CS. Cells were then stimulated with either EGF (275 ng/ml) or PDGF-BB (50 ng/ml) Intergert, Purchase, N. Y.) for 2 minutes in DMEM containing 1 mg/ml BSA and 20 mM HEPES pH 7.5, following which the cells were immediately washed and lysed.
  • DMEM Dulbecco's modified Eagles medium
  • CS calf serum
  • Lysate protein content was normalized as described (Bradford, 1976). Cell lysis, immunoprecipitation, and immunoblotting were performed as previously described (Margolis et al. Cell 57:1101-1107 (1989)). 293 cells were transfected using a modification of the calcium phosphate precipitation method (Chen and (Okayama Mol. Cell. Biol. 7:2745-272 (1987).
  • Monoclonal antiphosphotyrosine antibodies (1G2) covalently coupled to agarose were purchased from Oncogene Science (Manbasset, N. Y.). Anti-P-Tyr immunoblots were performed with a rabbit polyclonal antibody. Anti-EGF receptor immunoprecipitates were performed with monoclonal antibody mAb m108 (Bellot et al. J. Cell Biol. 110:491-502 (1990).
  • Anti-EGF receptor immunoblots were performed with anti-C terminus peptide (residues 1176-1186) antisera (Margolis et al. Cell 57:1101-1107 (1989)).
  • GST-GRB2 full length (FL) amino acids ⁇ AA ⁇ 2-217
  • GST-SH2 amino acids ⁇ AA ⁇ 2-217
  • GST-N-terminal SH3 AA 2-59
  • GST-C-terminal SH3 AA 156-217
  • GST-N-terminal SH3-SH2 AA-161
  • GST-SH22-C-terminal SH3 AA 50-217).
  • EGF EGF
  • PDGF PDGF
  • lysis buffer 10 mM Tris-Cl pH 7.6, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 100 uM sodium orthovanadate, 5 uM ZnCl 2 , 1 mM PMSF and 0.5% Triton-X-100.
  • the lysates where preclearea for 1 hour with 50 ul Sepharose G25, and then incubated overnight with anti-GRB2 antiserum (Ab50) at 4° C.
  • the immune complexes were then precipitated with protein A-Sepharose for 45 min at 4° C., washed 8-15 times with RIPA buffer (20 mM Tris-Cl pH 7.6, 300 mM NaCl, 2mM EDTA, 1% Triton-X-100, 1% sodium deoxycholate and 0.1% SDS), heated in Laemmli sample buffer containing 0.1 M B-mercaptoethanol and 1% SDS at 95° C.
  • RESULTS Isolation of a cDNA clone encoding a protein with novel SH2 and SH3 domains.
  • the carboxyterminal tail of the EGFR was used as a probe to screen a human brain stem ⁇ gt11 protein expression library as previously described (Skolnik et al. Cell 6:4396-4408, 1991).
  • One of the clones isolated utilizing this technique, clone 2-4 contained an insert of 1100 nucleotides found to contain a reading frame encoding novel SH2 and SH3 domains.
  • the insert from clone 2-4 contained a 3' stop codon followed by a polyadenylation signal, but did not contain the 5' start site.
  • the library was rescreened using DNA probes generated by amplifying DNA from clone 2-4.
  • GRB2 for the second growth factor receptor binding protein identified by the CORT method
  • the sequence also contains two potential protein kinase C phosphorylation sites (aa 22 and 102), two potential casein kinase 2 phosphorylation consensus sequences (aa 16 and 131) (Woodget et al. Eur. J. Biochem. 161:177-184 1986; Kishimoto et al. J. Biol. Chem.
  • GRB2 tissue distribution of GRB2
  • Northern hybridization analysis of various mouse tissue RNAs was performed, using as a probe the insert from clone 10-53. This analysis demonstrated GRB2 expression in every tissue examined, with the highest expression in the brain, spleen, lung, and intestine (FIG. 27A). GRB2 transcripts were visible in the thymus upon longer exposure. We have thus far been unable to identify a tissue or cell line which does not express GRB2, further demonstrating the ubiquitous nature of GRB2 expression. GRB2 hybridized to two transcripts of 1.5 and 3.8 kb. The 1.5 kb transcript corresponds to the expected size of clone 10-53.
  • FIG. 27B shows that a protein of 25 kDa is recognized by the immune, but not by the preimmune antiserumutilizing either immunoprecipitation analysis of ( 35 S) methionine labelled cells or an immunoblotting approach.
  • the various antisera recognized a 25 kDa protein in every cell line and tissue examined, consistent with the distribution of the GRB2 transcript found in Northern analysis.
  • GRB2 associates with growth factor receptors in living cells.
  • Receptor substrates which contain SH2 domains are endowed with the ability to physically associate with certain activated growth factor receptors. Since the goal of the CORT cloning technique is to identify target proteins for particular growth factor receptors, we assessed whether GRB2 associates with the EGF receptor.
  • HER 14 cells were treated with or without EGF, lysed, and subjected to immunoprecipitation analysis, according to published procedures (Margolis et al. 1990b, 1991b).
  • GRB2 associates only with the activated tyrosine phosphorylated EGFR.
  • GRB2 was also demonstrated to have an association with EGFR by immunoprecipitation of GRB2 followed by immunoblotting with anti EGF-receptor antibodies (data not shown). Similar results were obtained with PDGF receptor; activated PDGF receptor associated with GRB2 in HER14 cell sin growth factor dependent manner.
  • SH2 domains mediate the interaction of signalling molecules, such as PLC ⁇ or GAP, with tyrosine phosphorylated growth factor receptors (Koch et al. Science 252:668-674 (1991); Heldin et al. Trends in Biol. Sci. 16:450-452 (1991); Margolis et al. Cell Growth and Differentiation 3:73-80 (1992), Margolis et al. Nature 3556:71-74 1992).
  • signalling molecules such as PLC ⁇ or GAP
  • GRB2 binds to activated growth factor receptors without being phosphorylated in living cells.
  • GRB2 tyrosine phosphorylation was detected in 293 cells transiently overexpressing PDGFR and GRB2 as determined by anti-PTyr and anti-GRB2 blotting (data not shown).
  • a shift in the mobility of GRB2 was detected on anti-GRB2 (Ab86) blots, in the presence of activated PDGF receptor and the lower mobility form was shown to be tyrosine phosphorylated by anti-PTyr blotting.
  • Similar experiments have confirmed that the immunoprecipitating antibody (Ab50) will recognize tyrosine phosphorylated GRB2. This data suggest that it is possible to tyrosine phosphorylate GRB2 under conditions of overexpression of both receptor and GRB2 protein.
  • GRB2 represents the human homologue of the C. elegans gene product sem-5.
  • GRB2 is composed of one SH2 domain flanked by two SH3 domains in the order of SH3, SH2, SH3.
  • a C. elegans gene encoding for a protein with similar size and domain order has been cloned in the laboratory of R. Horvitz (Clark et al., 1992).
  • This gene, called sem-5 plays a crucial role in C. elegans development as mutations in sem-5 impair both vulval development and sex myoblast migration.
  • FIG. 32 shows a comparison of the amino acid sequences of GRB2 and sem-5.
  • the N-SH3 domains are 58% (63%) and the C-terminal SH3 domains are 58% identical (60%), respectively.
  • the overall sequence identity (similarity) is 58% (63%).
  • these two genes are very similar suggesting the sem-5 represents the C. elegans homologue of GRB2.
  • GRB2 A novel EGF receptor binding protein of the present invention was cloned by the CORT expression cloning method of the present invention, designated as GRB2.
  • This 25 kDa protein contains on SH2 domain and two SH3 domains.
  • GRB2 is widely expressed, as determined by Northern analysis in ten different murine tissues. It is also expressed in every human, monkey and murine cell line tested as revealed by Northern blotting, immunoprecipitation and immunoblotting experiments. Also shown is that GRB2 associates with EGF and PDGF receptors in a ligand-dependent manner, both in vitro and in living cells.
  • the association between GRB2 and growth factor receptors is mediated by the SH2 domain, can be dependent upon receptor tyrosine autophosphorylation, and involves a direct interaction between GRB2 and the tyrosine phosphorylated receptors.
  • GRB2 forms stable complexes with tyrosine phosphorylated, on tyrosine, serine, or threonine residues at physiologic levels of expression to any significant extent.
  • pretreatment of cells with vanadate did not increase GRB2 phosphorylation indicates that GRB2 is not rapidly dephosphorylated by tyrosine phosphatases.
  • let-60/ras functions downstream from EGFR and GRB2 and that GRB2 is somehow involved in regulation of ras activity.
  • the 55 kDa phosphoprotein which binds to GRB2 in response to growth factor stimulation is expected to be a downstream signaling molecule regulated upon GRB2 binding to activated growth factor receptors.
  • a T7 phage library expression system used an alternative to the phage ⁇ gt11 system described in Example II above, was used to express tyrosine kinase target proteins, as presented in the above Examples, with modifications as described below.
  • a T7 polymerase system (Palazzalo et al., Gene 88, 25 (1990); ⁇ EXlox vector, Novagert, Inc.), based on the PET expression systems of Studier and coworkers (Studier et al Meth. Enzymol. 185:60 (1990)) fusing cDNA clones to a fragment of the T7 capsid protein T10 under the control of the T7 promoter. These phages were then used to infect E.
  • the amplified DNA was cut with EcoR1 and ligated into EcoR1 digested ⁇ gt11 DNA (Promega). After packaging (Gigapack, Stragene), the phages were plated and screened with PLC- ⁇ 1 antibody using known techniques (Huynh, T. V. et al. In: DNA CLONING, ed. Glover, IRL Press, Oxford, 1:49-78 (1985)). This phage was then tested for binding to a cyanogen bromide generated fragment from 32 P-ATP labelled EGFR as described in the above Examples. An identical approach was taken to clone the two SH2 domains into ⁇ gt11 or ⁇ EXlox vectors.
  • FIG. 25 uniform binding of the EGFR was seen in the that appeared stronger than was seen with the ⁇ gt11 system (compare FIG. 25A and 25B).
  • FIG. 25A and 25B We also cloned in a longer fragment which ran from 532-1290 of PLC ⁇ 1 and this was also easily seen in the T7 system (FIG. 25).
  • the T7 plaques although mostly smaller than the ⁇ gt11 plaques gave stronger signals. This makes this system particularly suitable for library screening when there as thousands of small plaques per plate.
  • the major advantage of this system is the high level of protein expression due to the greater activity of the T7 polymerase versus E. coli RNA polymerase.
  • the fusion proteins using the smaller T10 gene fragment yields more stable expression and that its hydrophobic character promotes binding to nitrocellulose.
  • the ⁇ EXlox phages also allow for automatic conversion to a PET plasmid (Palazzalo et al., Gene 88, 25 (1990)) which can be useful for expression of a fusion protein for antibody production. Accordingly, screening an T7 expression library is expected to give superior results than for ⁇ gt11 for such a cloning strategy of the present invention.
  • GRB-3 has a high degree of identity with v-crk beginning with the methionine at residue 32 and this methionine has been found to be the start site of arian c-crk.
  • this methionine has been found to be the start site of arian c-crk.
  • GRB-4 was similar to nck (FIG. 18), a human protein composed of three SH3 domains and one SH2 domain.
  • Our clone contained one SH3 domain and one SH2 domain and was 74% identical at the protein level and 66% similar at the DNA level in the open reading frame.
  • the T7 ( ⁇ EXlox) library was plated in an E. coli strain without T7 polymerase gene and routine DNA hybridization performed with a 700 base pair EcoR1 fragment from the GRB-7 clone using standard published techniques (Ausubel et al eds., Current Protocols in Molecular Biology, Wiley interscience, New York, (1987, 1992)).
  • Several overlapping clones were identified which were used for DNA sequencing to obtain the full length GRB-7 protein sequence shown in FIG. 19.
  • a schematic representation of GRB-7 is displayed in FIG. 20 depicting the regions of similarity to known proteins as discussed below.
  • the protein is 535 amino acids in length and has one SH2 domain at its extreme carboxy-terminus.
  • the SH2 domain of GRB-7 is compared to other SH2 domains including mouse fyn, human PLC- ⁇ 1 and the crk and nck-like proteins we cloned in this project.
  • GRB-7 has an isoleucine at amino acid 448, whereas other SH2 domains have a leucine at this position.
  • a sequence of 433 amino acids from GRB-7 which excluded the SH2 domain was used to scan the Swissprot and GenEmbl databases, as described herein.
  • Amino acids 242 to 339 of GRB-7 showed similarity to a sequence from the central region of ras GAP. Over this region of 91 amino acids from ras GAP, GRB-7 has 26% identity and 42% similarity allowing for conservative substitutions (FIG. 22). This region of ras GAP lies between the SH2/SH3 domains and he GTPase activating carboxyterminal region and has not been assigned a specific function. The amino-terminal sequence of GRB-7 was found to be proline rich and thus has similarity to many other proline rich proteins. GRB-7 does have an extended region of limited similarity to the catalytic domain of protein phosphatase 2B including this proline rich region (FIG. 23) but no significant similarity was found to other serine/threonine phosphatase such as protein phosphatase 1 or 2A.
  • FIG. 24 A northern blot of GRB-7 in mouse tissues is presented in FIG. 24. Oligo dt selected mRNA was probed with the same EcoR1 fragment used to isolate full length GRB-7. See Ausubel et al eds., Current Protocols in Molecular Biology, Wiley Interscience, New York, (1987, 1992) and Sap et al Proc. Natl. Acad. Sci. USA 87:6112 (1990). The mRNA was extracted from six week old mice tissues by known methods, e.g., as described by Sap et al Proc. Natl. Acad. Sci. USA 87:6112 (1990).
  • Example IX presents the cloning, via the CORT method, and characterization of the GRB-10 gene.
  • the GRB-10 gene exhibits a high level of homology to the GRB-7 gene. Such homology indicates that GRB-10 and GRB-7 represent a family of genes likely to have overlapping functions.
  • GRB-10 was cloned from a ⁇ EXlox NIH 3T3 (mouse fibroblast cell line) using the CORT technique, as described in the Detailed Description of the Preferred Embodiments, above.
  • the probe utilized was the EGF-Receptor carboxyterminus.
  • the randomly primed NIH 3T3 library was generated using standard techniques (Sambrook et al. 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor). After the initial clone was isolated, GRB-10 cDNA encoding the full length GRB-7 protein was cloned from the same library using DNA hybridization as described (Margolis et al. 1992, Proc. Natl. Acad. Sci.
  • FIG. 37A-B The cDNA sequence is presented in FIG. 37A-B and the protein sequence in FIG. 38A-E.
  • FIG. 39 combines protein and cDNA data.
  • the GRB-10 protein is highly related to the GRB-7 protein with an overall amino acid identity of 51% (FIG. 40A-40B).
  • the major regions of similarity are schematically depicted in FIG. 41 and primarily consist of the carboxyterminal SH2 domain and a larger central domain. They also share a common central domain of approximately 330 amino acids with an identity of 54%. This central domain is also found in one other protein in the Genbank database.
  • This gene known as FlOE9.6, was identified by the Caenorhabditis Eleqans genome sequencing project during sequencing of C. Elegans chromosome III. It is noteworthy that FLOE9.6 does not contain an SH2 domain but does contain a proline rich domain as do GRB-7 and GRB-10.
  • FIG. 42 The amino acid alignment of the GRB-10 SH2 domain with SH2 domains from GRB-7, GRB-2 and c-SRC is shown in FIG. 42.
  • FIG. 43 displays the amino acid alignment of the central domains and includes a domain found in the Caenorhabditis Elegans gene, FLOE9.6, a gene identified by the C. Elegans genome sequencing project (Sulston et al. 1992, Nature 356: 37-41). This C. Elegans gene is also schematically depicted in FIG. 41.
  • the blot was probed with a 32 P-dCTP labeled fragment of GRB-10.
  • the membrane was subject to prehybridization (4 hours) and hybridization (overnight) in the Church buffer (7% SDS, 1% BSA, 1 mM EDTA, 250 mM Na 2 HPO 4 , pH 7.2) at 60° C.
  • the next day the blots were washed with high stringency buffer (40 mM sodium phosphate, pH 7.2, 1% SDS, 1 mM EDTA) at 60° C.
  • the blot was stripped and reprobed with actin (bottom).
  • the mRNA from lung was degraded but GRB-10 message could be detected in total RNA.
  • the GRB-10 protein is also detected in NIH 3T3 fibroblast cells, rat L6 skeletal muscle cells, rat mesangial cells and dog kidney MDCK epithelial cells.
  • GRB-10 The spatial expression pattern of GRB-10 contrasts with that seen for GRB-7, with GRB-7 found only in liver, kidney and testes.
  • the results indicate that GRB-7 and GRB-10 represent a family of Series that are likely to have overlapping functions but individual patterns of expression.

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PCT/US1995/003385 WO1995024426A1 (fr) 1994-03-11 1995-03-13 Nouveau procede d'expression-clonage utilise pour identifier des proteines a cibles des tirosine-kinases eukaryotes, et nouvelles proteines cibles
CA002184988A CA2184988A1 (fr) 1994-03-11 1995-03-13 Nouvelle methode d'expression-clonage pour caracteriser les proteines cibles pour tyrosines-kinases eucaryotes; nouvelles proteines cibles
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US07/906,349 US5434064A (en) 1991-01-18 1992-06-30 Expression-cloning method for identifying target proteins for eukaryotic tyrosine kinases and novel target proteins
US08/167,035 US5618691A (en) 1991-01-18 1993-12-16 Recombinant DNA encoding a eukaryotic tyrosine kinase target protein
US08/208,887 US5677421A (en) 1991-01-18 1994-03-11 Target proteins for eukaryotic tyrosine kinases

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CA2184988A1 (fr) 1995-09-14

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